Method to identify asian soybean rust resistance quantitative trait loci in soybean and compositions thereof

ABSTRACT

The present invention is in the field of plant breeding and disease resistance. More specifically, the invention includes a method for breeding soybean plants containing quantitative trait loci that are associated with resistance to Asian Soybean Rust (ASR), a fungal disease associated with  Phakopsora  spp. The invention further includes germplasm and the use of germplasm containing quantitative trait loci (QTL) conferring disease resistance for introgression into elite germplasm in a breeding program for resistance to ASR.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit under 35 U.S.C. §119(e) of U.S.Provisional Application No. 61/047,479 filed 24 Apr. 2008. The entiretyof the application is hereby incorporated by reference.

INCORPORATION OF THE SEQUENCE LISTING

A sequence listing containing the file named “pa_(—)54987b.txt” which is25,543 bytes (measured in Microsoft Windows®) and created on April6^(th), 2009, comprises 80 nucleotide sequences, and is hereinincorporated by reference in its entirety.

FIELD OF THE INVENTION

The present invention is in the field of plant breeding and diseaseresistance. More specifically, the invention includes a method forbreeding soybean plants containing quantitative trait loci that areassociated with resistance to Asian soybean rust disease (ASR) caused byPhakopsora pachyrhizi and Phakopsora meibomiae. The invention furtherincludes germplasm, novel quantitative trait loci (QTL) conferringresistance to ASR, and methods for introgressing the novel QTL intoelite germplasm in a breeding program for resistance to ASR.

BACKGROUND OF THE INVENTION

The soybean, Glycine max (L.) Merril, is one of the major economic cropsgrown worldwide as a primary source of vegetable oil and protein(Sinclair and Backman, Compendium of Soybean Diseases, 3^(rd) Ed. APSPress, St. Paul, Minn., p. 106 (1989)). The growing demand for lowcholesterol and high fiber diets has also increased soybean's importanceas a health food.

Soybean yields in the United States are negatively affected each year bydiseases. High yields per hectare are critical to a farmer's profitmargin, especially during periods of low prices for soybean. Thefinancial loss caused by soybean diseases is important to ruraleconomies and to the economies of allied industries in urban areas. Theeffects of these losses are eventually felt throughout the soybeanmarket worldwide.

Asian Soybean Rust (herein referred to as ASR) has been reported in theEastern and Western Hemispheres. In the Eastern Hemisphere, ASR has beenreported in Australia, China, India, Japan, Taiwan and Thailand. In theWestern Hemisphere, ASR has been observed in Brazil, Columbia, CostaRica and Puerto Rico. ASR can be a devastating disease, causing yieldlosses of up to 70 to 80% as reported in some fields in Taiwan. Plantsthat are heavily infected have fewer pods and smaller seeds that are ofpoor quality (Frederick et al., Mycology 92: 217-227 (2002)). ASR wasfirst observed in the United States in Hawaii in 1994. ASR was laterintroduced into the continental United States in the fall of 2004,presumably as a consequence of tropical storm activity. Modelpredictions indicated that ASR had been widely dispersed throughout thesoutheastern United States, and subsequent field and laboratoryobservations confirmed this distribution.

Two species of fungi, Phakopsora pachyrhizi Sydow and Phakopsorameibomiae (Arthur) Arthur, cause ASR. Unlike other rusts, P. pachyrhiziand P. meibomiae infect an unusually broad range of plant species. P.pachyrhizi is known to naturally infect 31 species in 17 genera oflegumes and 60 species in 26 other genera have been infected undercontrolled conditions. P. meibomiae naturally infects 42 species in 19genera of legumes, and 18 additional species in 12 other genera havebeen artificially infected. Twenty-four plant species in 19 genera arehosts for both species (Frederick et al., Mycology 92: 217-227 (2002)).

Evaluating plants that could potentially contain QTL conferringresistance to ASR can be time consuming and require large amounts ofbiologically contained space. Culturing P. pachyrhizi requires the useof an approved biological containment hood. In addition, greenhouses andgrowth chambers used to grow plants for ASR resistance testing have tobe constructed in a manner that prevents the accidental release of theorganism, especially in locations in which the organism has still notyet been observed. Different cultures of P. pachyrhizi may possessdifferent virulence factors. Over time, new strains of P. pachyrhizi maybe introduced into the United States. The two principal hosts are yambean (Pachyrhizus erosus (L.) Urban) and cowpea (Vigna unguiculata (L.)Walp.), both found in Florida. One widespread naturalized host for P.pachyrhizi and P. meibomiae is kudzu (Pueraria montana (Lour.) Men. var.lobata (Willd.) Maesen & S. M. Almeida ex Sanjappa & Predeep). Becausekudzu is a common weed in the southeastern United States, it might serveas a continual source of inoculum. Both P. pachyrhizi and P. meibomiaeare autoecious (no alternate hosts) and microcyclic (with uredinial andtelial spore stages), with the obligate pathogens surviving andreproducing only on live hosts. Additional hosts can serve asover-wintering reservoirs for the pathogen, as well as build-up ofinoculum. The pathogen is well adapted for long-distance dispersal,because the spores can be readily carried by the wind, making it anideal means for introduction to new, rust-free regions. The primarymeans of dissemination are spores, which can be carried by wind orsplashed rain.

Because different cultures of P. pachyrhizi may possess differentvirulence factors to known and suspected genes for resistance, itfollows that different ASR resistance loci in the soybean genome may beexpected to differ with respect to which strain(s) of P. pachyrhizi andor P. meibomiae they confer resistance against. Therefore, any breedingprogram designed to breed resistance into soybean against ASR willlikely need to involve multifactorial resistance derived from differentresistance loci in the soybean genome, in order to confer robustresistance against ASR despite changes in the P. pachyrhizi population.Also, breeding for soybean crops used in other geographic locations willrequire selecting resistance to the specific strains that affect thoseregions, in addition to providing those agronomic characteristics thatare preferred by these farmers in that region. There is therefore strongmotivation to identify novel ASR resistance loci in soybean, and tointrogress desirable alleles into elite soybean germplasm. Methodologyhas been developed to evaluate plants that could potentially contain QTLconferring resistance to ASR (U.S. Patent Appl No. 20080166699).

SUMMARY OF THE INVENTION

The present invention provides a method for introgressing an allele intoa soybean plant comprising: crossing at least one ASR resistant soybeanplant with at least one other soybean plant to form a population; andscreening the population with at least one nucleic acid marker from thegroup consisting of ASR resistance loci 14, 15 and 16, to determine ifone or more soybean plants from the population contains at least one ASRresistance allele from the group consisting of ASR resistance alleles 1through 8. In various embodiments, the at least one marker is locatedwithin 30 cM, 15 cM, 5 cM or 1 cM of the resistance allele, or within 1Mb, 100 Kb or 1 Kb of the resistance allele.

In another aspect, the invention provides an elite soybean plantproduced by: crossing at least one ASR resistant soybean plant with atleast one other soybean plant to form a population; and screening thepopulation with at least one nucleic acid marker from the groupconsisting of ASR resistance loci 14, 15 and 16, to determine if one ormore soybean plants from the population contains at least one ASRresistance allele from the group consisting of ASR resistance alleles 1through 8. In one embodiment, the elite soybean plant exhibits at leastone transgenic trait. In a more particular embodiment, the at least onetransgenic trait may be herbicide tolerance, increased yield, insectcontrol, fungal disease resistance, virus resistance, nematoderesistance, bacterial disease resistance, mycoplasma disease resistance,modified oils production, high oil production, high protein production,germination and seedling growth control, enhanced animal and humannutrition, low raffinose, environmental stress resistance, increaseddigestibility, improved processing traits, improved flavor, nitrogenfixation, hybrid seed production, reduced allergenicity or anycombination thereof. In a yet more particular embodiment, herbicidetolerance may be conferred for glyphosate, dicamba, glufosinate,sulfonylurea, bromoxynil, 2,4-Dichlorophenoxyacetic acid, norflurazonherbicides or any combination thereof. In yet another embodiment, theelite soybean plant exhibits at least partial resistance to at least onerace of an ASR-inducing fungus and more particularly the ASR-inducingfungus may be Phakopsora. pachyrhizi or P. meibomiae or both.

The invention also provides a method of introgressing at least one ASRresistance allele into a soybean plant comprising the steps: crossing anASR resistant soybean plant with a second soybean plant to form apopulation; screening the population with at least one nucleic acidmarker selected from the group consisting of SEQ ID NO: 1 through 8 andSEQ ID NO: 73 through 80; and selecting from the population at least onesoybean plant comprising at least one genotype corresponding to the ASRresistant soybean plant. In particular embodiments, the selected soybeanplant exhibits a resistant reaction rating to ASR of no worse than about3, or of no worse than about 2, as described herein. In a moreparticular embodiment, the method further comprises the step of assayingthe selected soybean plant for resistance to an ASR inducing pathogen.In another particular embodiment, the genotype is determined by singlebase extension (SBE), allele-specific primer extension sequencing(ASPE), DNA sequencing, RNA sequencing, microarray-based analyses,universal PCR, allele specific extension, hybridization, massspectrometry, ligation, extension-ligation, or FlapEndonuclease-mediated assays. In still more particular embodiments, themethod further comprises the step of crossing the selected soybean plantto another soybean plant; and still further comprises the step ofobtaining seed from the selected soybean plant. In yet anotherparticular embodiment, the at least one soybean plant in the populationis genotyped with respect to a soybean genomic DNA marker selected fromthe group consisting of SEQ ID NO: 1 and 2 and with respect to SEQ IDNO: 3.

In another aspect, the invention provides an elite soybean plantproduced by:

crossing an ASR resistant soybean plant with a second soybean plant toform a population; screening said population with at least one nucleicacid marker selected from the group consisting of SEQ ID NO: 1 through8, and SEQ ID NO: 73 through 80; and selecting from said population oneor more soybean plants comprising at least one genotype corresponding tothe ASR resistant soybean plant. In one embodiment, the elite soybeanplant exhibits at least one transgenic trait. In a more particularembodiment, the transgenic trait may be herbicide tolerance, increasedyield, insect control, fungal disease resistance, virus resistance,nematode resistance, bacterial disease resistance, mycoplasma diseaseresistance, modified oils production, high oil production, high proteinproduction, germination and seedling growth control, enhanced animal andhuman nutrition, low raffinose, environmental stress resistance,increased digestibility, improved processing traits, improved flavor,nitrogen fixation, hybrid seed production, reduced allergenicity or anycombination thereof. In a yet more particular embodiment, herbicidetolerance may be conferred for glyphosate, dicamba, glufosinate,sulfonylurea, bromoxynil, 2,4-Dichlorophenoxyacetic acid, norflurazonherbicides or any combination thereof. In yet other embodiments, theelite soybean plant exhibits at least partial resistance to at least onerace of an ASR-inducing fungus and more particularly the ASR-inducingfungus may be Phakopsora pachyrhizi or Phakopsora meibomiae or both.

The invention also provides a substantially purified nucleic acidmolecule for the detection of loci related to ASR resistance, comprisinga nucleic acid sequence selected from the group consisting of SEQ ID NO:1 through SEQ ID NO: 80 and complements thereof.

Further, the invention provides an isolated nucleic acid molecule of atleast 15, 16, 17, 18 or 20 nucleotides, having at least 90% identity toa sequence of the same length in either strand of soybean DNA thatincludes or is adjacent to a polymorphism, for detecting a molecularmarker representing the polymorphism, wherein the molecular marker isselected from the group consisting of SEQ ID NO: 1 though 8. In anotherembodiment, the invention provides an isolated nucleic acid molecule ofat least 15, 16, 17, 18 or 20 nucleotides, having at least 95%, orpreferably 98%, or more preferably 99% or even 100% identity to asequence of the same length in either strand of soybean DNA thatincludes or is adjacent to a polymorphism, for detecting a molecularmarker representing the polymorphism, wherein the molecular marker isselected from the group consisting of SEQ ID NO: 1 though 8. In aparticular embodiment, the isolated nucleic acid further comprises adetectable label or provides for incorporation of a detectable label.More particularly, the detectable label may be an isotope, afluorophore, an oxidant, a reductant, a nucleotide or a hapten. In a yetmore particular embodiment, the detectable label may be added to thenucleic acid by a chemical reaction or incorporated by an enzymaticreaction. In another embodiment of the invention, the isolated nucleicacid hybridizes to at least one allele of the molecular marker understringent hybridization conditions. In more particular embodiments, themolecular marker is SEQ ID NO: 1, 2, 3, 4, 5, 6, 7 or 8; and theisolated nucleic acid is an oligonucleotide that is at least 90%identical to provided probes corresponding to the particular molecularmarker, which are respectively: SEQ ID NO: 25 and 26, 27 and 28, 29 and30, 31 and 32, 33 and 34, and 36, 37 and 38, or 39 and 40.

The invention also provides a set of oligonucleotides comprising: a pairof oligonucleotide primers, each at least 12 contiguous nucleotideslong, that permit PCR amplification of a DNA segment comprising orcontained within a molecular marker selected from the group consistingof SEQ ID NO: 1 through 8; and at least one detector oligonucleotidethat permits detection of a polymorphism in the amplified segment,wherein the sequence of the detector oligonucleotide is at least 95percent identical to a sequence of the same number of consecutivenucleotides in either strand of a segment of soybean DNA that include orare adjacent to the polymorphism. In one embodiment, the detectoroligonucleotide comprises at least 12 nucleotides and either providesfor incorporation of a detectable label or further comprises adetectable label. In a more particular embodiment, the detectable labelis selected from the group consisting of an isotope, a fluorophore, anoxidant, a reductant, a nucleotide and a hapten. In another embodiment,the detector oligonucleotide and said oligonucleotide primers hybridizeto at least one allele of the molecular marker under stringenthybridization conditions. Yet other embodiments comprise a seconddetector oligonucleotide capable of detecting a distinct secondpolymorphism of the molecular marker or a distinct allele of the samepolymorphism. In yet more particular embodiments, the molecular markeris SEQ ID NO: 1, 2, 3, 4, 5, 6, 7 or 8; and the oligonucleotide primersare at least 90% identical to provided primers corresponding to theparticular molecular marker, which are respectively: SEQ ID NO: 9 and10, 11 and 12, 13 and 14, 15 and 16, 17 and 18, 19 and 20, 21 and 22, or23 and 24; and the detector oligonucleotide comprises a nucleic acidthat is at least 90% identical to provided probes corresponding to theparticular molecular marker, which are respectively: SEQ ID NO: 25 and26, 27 and 28, 29 and 30, 31 and 32, 33 and 34, 35 and 36, 37 and 38, or39 and 40.

The invention also provides a method of introgressing an allele into asoybean plant comprising: providing a population of soybean plants;genotyping at least one soybean plant in the population with respect toa soybean genomic nucleic acid marker selected from the group SEQ ID NO:1 to 8 and SEQ ID NO: 73 to 80; and selecting from the population one ormore soybean plants comprising an allele associated with ASR resistance,wherein said ASR resistance allele is selected from the group consistingof SEQ ID NO: 73 through SEQ ID NO: 80. In one embodiment, providing apopulation comprises crossing an ASR resistant soybean plant with asecond soybean plant to form a population. In another embodiment, theselected one or more soybean plants exhibit increased grain yield in thepresence of ASR as compared to soybean plants lacking ASR resistancealleles. More particularly, increased grain yield may be at least 0.5Bu/A, at least 1.0 Bu/A, or at least 1.5 Bu/A in the presence of ASR ascompared to soybean plants lacking ASR resistance alleles.

BRIEF DESCRIPTION OF NUCLEIC ACID SEQUENCES

SEQ ID NO: 1 is a genomic sequence derived from Glycine max associatedwith ASR resistance locus 14.

SEQ ID NO: 2 is a genomic sequence derived from Glycine max associatedwith ASR resistance locus 14.

SEQ ID NO: 3 is a genomic sequence derived from Glycine max associatedwith ASR resistance locus 14.

SEQ ID NO: 4 is a genomic sequence derived from Glycine max associatedwith ASR resistance locus 15.

SEQ ID NO: 5 is a genomic sequence derived from Glycine max associatedwith ASR resistance locus 15.

SEQ ID NO: 6 is a genomic sequence derived from Glycine max associatedwith ASR resistance locus 15.

SEQ ID NO: 7 is a genomic sequence derived from Glycine max associatedwith ASR resistance locus 16.

SEQ ID NO: 8 is a genomic sequence derived from Glycine max associatedwith ASR resistance locus 16.

SEQ ID NO: 9 is a forward PCR primer for the amplification of SEQ ID NO:1.

SEQ ID NO: 10 is a reverse PCR primer for the amplification of SEQ IDNO: 1.

SEQ ID NO: 11 is a forward PCR primer for the amplification of SEQ IDNO: 2.

SEQ ID NO: 12 is a reverse PCR primer for the amplification of SEQ IDNO: 2.

SEQ ID NO: 13 is a forward PCR primer for the amplification of SEQ IDNO: 3.

SEQ ID NO: 14 is a reverse PCR primer for the amplification of SEQ IDNO: 3.

SEQ ID NO: 15 is a forward PCR primer for the amplification of SEQ IDNO: 4.

SEQ ID NO: 16 is a reverse PCR primer for the amplification of SEQ IDNO: 4.

SEQ ID NO: 17 is a forward PCR primer for the amplification of SEQ IDNO: 5.

SEQ ID NO: 18 is a reverse PCR primer for the amplification of SEQ IDNO: 5.

SEQ ID NO: 19 is a forward PCR primer for the amplification of SEQ IDNO: 6.

SEQ ID NO: 20 is a reverse PCR primer for the amplification of SEQ IDNO: 6.

SEQ ID NO: 21 is a forward PCR primer for the amplification of SEQ IDNO: 7.

SEQ ID NO: 22 is a reverse PCR primer for the amplification of SEQ IDNO: 7.

SEQ ID NO: 23 is a forward PCR primer for the amplification of SEQ IDNO: 8.

SEQ ID NO: 24 is a reverse PCR primer for the amplification of SEQ IDNO: 8.

SEQ ID NO: 25 is a probe for the detection of the SNP of SEQ ID NO: 1.

SEQ ID NO: 26 is a probe for the detection of the SNP of SEQ ID NO: 1.

SEQ ID NO: 27 is a probe for the detection of the SNP of SEQ ID NO: 2.

SEQ ID NO: 28 is a probe for the detection of the SNP of SEQ ID NO: 2.

SEQ ID NO: 29 is a probe for the detection of the SNP of SEQ ID NO: 3.

SEQ ID NO: 30 is a probe for the detection of the SNP of SEQ ID NO: 3.

SEQ ID NO: 31 is a probe for the detection of the SNP of SEQ ID NO: 4.

SEQ ID NO: 32 is a probe for the detection of the SNP of SEQ ID NO: 4.

SEQ ID NO: 33 is a probe for the detection of the SNP of SEQ ID NO: 5.

SEQ ID NO: 34 is a probe for the detection of the SNP of SEQ ID NO: 5.

SEQ ID NO: 35 is a probe for the detection of the SNP of SEQ ID NO: 6.

SEQ ID NO: 36 is a probe for the detection of the SNP of SEQ ID NO: 6.

SEQ ID NO: 37 is a probe for the detection of the SNP of SEQ ID NO: 7.

SEQ ID NO: 38 is a probe for the detection of the SNP of SEQ ID NO: 7.

SEQ ID NO: 39 is a probe for the detection of the SNP of SEQ ID NO: 8.

SEQ ID NO: 40 is a probe for the detection of the SNP of SEQ ID NO: 8.

SEQ ID NO: 41 is a hybridization probe for an ASR resistance allelecorresponding to SEQ ID NO: 1.

SEQ ID NO: 42 is a hybridization probe for an ASR susceptibility allelecorresponding to SEQ ID NO: 1.

SEQ ID NO: 43 is a hybridization probe for an ASR resistance allelecorresponding to SEQ ID NO: 2.

SEQ ID NO: 44 is a hybridization probe for an ASR susceptibility allelecorresponding to SEQ ID NO: 2.

SEQ ID NO: 45 is a hybridization probe for an ASR resistance allelecorresponding to SEQ ID NO: 3.

SEQ ID NO: 46 is a hybridization probe for an ASR susceptibility allelecorresponding to SEQ ID NO: 3.

SEQ ID NO: 47 is a hybridization probe for an ASR resistance allelecorresponding to SEQ ID NO: 4.

SEQ ID NO: 48 is a hybridization probe for an ASR susceptibility allelecorresponding to SEQ ID NO: 4.

SEQ ID NO: 49 is a hybridization probe for an ASR resistance allelecorresponding to SEQ ID NO: 5.

SEQ ID NO: 50 is a hybridization probe for an ASR susceptibility allelecorresponding to SEQ ID NO: 5.

SEQ ID NO: 51 is a hybridization probe for an ASR resistance allelecorresponding to SEQ ID NO: 6.

SEQ ID NO: 52 is a hybridization probe for an ASR susceptibility allelecorresponding to SEQ ID NO: 6.

SEQ ID NO: 53 is a hybridization probe for an ASR resistance allelecorresponding to SEQ ID NO: 7.

SEQ ID NO: 54 is a hybridization probe for an ASR susceptibility allelecorresponding to SEQ ID NO: 7.

SEQ ID NO: 55 is a hybridization probe for an ASR resistance allelecorresponding to SEQ ID NO: 8.

SEQ ID NO: 56 is a hybridization probe for an ASR susceptibility allelecorresponding to SEQ ID NO: 8.

SEQ ID NO: 57 is a single base extension (SBE) probe for an ASRresistance allele corresponding to SEQ ID NO: 1.

SEQ ID NO: 58 is a single base extension (SBE) probe for an ASRresistance allele corresponding to SEQ ID NO: 1.

SEQ ID NO: 59 is a single base extension (SBE) probe for an ASRresistance allele corresponding to SEQ ID NO: 2.

SEQ ID NO: 60 is a single base extension (SBE) probe for an ASRresistance allele corresponding to SEQ ID NO: 2.

SEQ ID NO: 61 is a single base extension (SBE) probe for an ASRresistance allele corresponding to SEQ ID NO: 3.

SEQ ID NO: 62 is a single base extension (SBE) probe for an ASRresistance allele corresponding to SEQ ID NO: 3.

SEQ ID NO: 63 is a single base extension (SBE) probe for an ASRresistance allele corresponding to SEQ ID NO: 4.

SEQ ID NO: 64 is a single base extension (SBE) probe for an ASRresistance allele corresponding to SEQ ID NO: 4.

SEQ ID NO: 65 is a single base extension (SBE) probe for an ASRresistance allele corresponding to SEQ ID NO: 5.

SEQ ID NO: 66 is a single base extension (SBE) probe for an ASRresistance allele corresponding to SEQ ID NO: 5.

SEQ ID NO: 67 is a single base extension (SBE) probe for an ASRresistance allele corresponding to SEQ ID NO: 6.

SEQ ID NO: 68 is a single base extension (SBE) probe for an ASRresistance allele corresponding to SEQ ID NO: 6.

SEQ ID NO: 69 is a single base extension (SBE) probe for an ASRresistance allele corresponding to SEQ ID NO: 7.

SEQ ID NO: 70 is a single base extension (SBE) probe for an ASRresistance allele corresponding to SEQ ID NO: 7.

SEQ ID NO: 71 is a single base extension (SBE) probe for an ASRresistance allele corresponding to SEQ ID NO: 8.

SEQ ID NO: 72 is a single base extension (SBE) probe for an ASRresistance allele corresponding to SEQ ID NO: 8.

SEQ ID NO: 73 is ASR resistance allele 1 corresponding to SEQ ID NO: 1.

SEQ ID NO: 74 is ASR resistance allele 2 corresponding to SEQ ID NO: 2.

SEQ ID NO: 75 is ASR resistance allele 3 corresponding to SEQ ID NO: 3.

SEQ ID NO: 76 is ASR resistance allele 4 corresponding to SEQ ID NO: 4.

SEQ ID NO: 77 is ASR resistance allele 5 corresponding to SEQ ID NO: 5.

SEQ ID NO: 78 is ASR resistance allele 6 corresponding to SEQ ID NO: 6.

SEQ ID NO: is an ASR resistance allele 7 corresponding to SEQ ID NO: 7.

SEQ ID NO: 80 is ASR resistance allele 8 corresponding to SEQ ID NO: 8.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the positions of ASR locus 14 on linkage group G. To theright is the legend for the LOD plot for the population. The black barindicates the confidence interval for the position of NS0102630(LOD>10). The gray bar indicates the confidence interval for theposition of NS0119675 (LOD>2).

FIG. 2 depicts the positions of ASR locus 15 on linkage group C2. To theright is the legend for the LOD plot for the population. The black barindicates the confidence interval for the position of NS0093385(LOD>10). The dark gray bar indicates the confidence interval for theposition of NS0118716 (LOD>2).

FIG. 3 depicts the positions of ASR locus 16 on linkage group D2. To theright is the legend for the LOD plot for the population. The gray barindicates the confidence interval for the position of NS0113966 (LOD>2).

DETAILED DESCRIPTION OF THE INVENTION

The definitions and methods provided define the present invention andguide those of ordinary skill in the art in the practice of the presentinvention. Unless otherwise noted, terms are to be understood accordingto conventional usage by those of ordinary skill in the relevant art.Definitions of common terms in molecular biology may also be found inAlberts et al., Molecular Biology of The Cell, 5^(th) Edition, GarlandScience Publishing, Inc.: New York, 2007; Rieger et al., Glossary ofGenetics: Classical and Molecular, 5th edition, Springer-Verlag: NewYork, 1991; King et al, A Dictionary of Genetics, 6th ed, OxfordUniversity Press: New York, 2002; and Lewin, Genes IX, Oxford UniversityPress: New York, 2007. The nomenclature for DNA bases as set forth at 37CFR §1.822 is used.

An “allele” refers to an alternative sequence at a particular locus; thelength of an allele can be as small as 1 nucleotide base, but istypically larger. Allelic sequence can be denoted as nucleic acidsequence or as amino acid sequence that is encoded by the nucleic acidsequence.

A “locus” is a position on a genomic sequence that is usually found by apoint of reference; e.g., a short DNA sequence that is a gene, or partof a gene or intergenic region. The loci of this invention comprise oneor more polymorphisms in a population; i.e., alternative alleles presentin some individuals.

As used herein, “polymorphism” means the presence of one or morevariations of a nucleic acid sequence at one or more loci in apopulation of one or more individuals. The variation may comprise but isnot limited to one or more base changes, the insertion of one or morenucleotides or the deletion of one or more nucleotides. A polymorphismmay arise from random processes in nucleic acid replication, throughmutagenesis, as a result of mobile genomic elements, from copy numbervariation and during the process of meiosis, such as unequal crossingover, genome duplication and chromosome breaks and fusions. Thevariation can be commonly found, or may exist at low frequency within apopulation, the former having greater utility in general plant breedingand the latter may be associated with rare but important phenotypicvariation. Useful polymorphisms may include single nucleotidepolymorphisms (SNPs), insertions or deletions in DNA sequence (Indels),simple sequence repeats of DNA sequence (SSRs) a restriction fragmentlength polymorphism, and a tag SNP. A genetic marker, a gene, aDNA-derived sequence, a haplotype, a RNA-derived sequence, a promoter, a5′ untranslated region of a gene, a 3′ untranslated region of a gene,microRNA, siRNA, a QTL, a satellite marker, a transgene, mRNA, ds mRNA,a transcriptional profile, and a methylation pattern may comprisepolymorphisms.

As used herein, “marker” means a polymorphic nucleic acid sequence ornucleic acid feature. A marker may be represented by one or moreparticular variant sequences, or by a consensus sequence. In anothersense, a “marker” is an isolated variant or consensus of such asequence. In a broader aspect, a “marker” can be a detectablecharacteristic that can be used to discriminate between heritabledifferences between organisms. Examples of such characteristics mayinclude genetic markers, protein composition, protein levels, oilcomposition, oil levels, carbohydrate composition, carbohydrate levels,fatty acid composition, fatty acid levels, amino acid composition, aminoacid levels, biopolymers, pharmaceuticals, starch composition, starchlevels, fermentable starch, fermentation yield, fermentation efficiency,energy yield, secondary compounds, metabolites, morphologicalcharacteristics, and agronomic characteristics.

As used herein, “marker assay” means a method for detecting apolymorphism at a particular locus using a particular method, e.g.measurement of at least one phenotype (such as seed color, flower color,or other visually detectable trait), restriction fragment lengthpolymorphism (RFLP), single base extension, electrophoresis, sequencealignment, allelic specific oligonucleotide hybridization (ASO), randomamplified polymorphic DNA (RAPD), microarray-based technologies, andnucleic acid sequencing technologies, etc.

As used herein, “typing” refers to any method whereby the specificallelic form of a given soybean genomic polymorphism is determined. Forexample, a single nucleotide polymorphism (SNP) is typed by determiningwhich nucleotide is present (i.e. an A, G, T, or C). Insertion/deletions(Indels) are determined by determining if the Indel is present. Indelscan be typed by a variety of assays including, but not limited to,marker assays.

As used herein, the phrase “adjacent”, when used to describe a nucleicacid molecule that hybridizes to DNA containing a polymorphism, refersto a nucleic acid that hybridizes to DNA sequences that directly ornearly abut the polymorphic nucleotide base position. For example, anucleic acid molecule that can be used in a single base extension assayis “adjacent” to the polymorphism.

As used herein, “interrogation position” refers to a physical positionon a solid support that can be queried to obtain genotyping data for oneor more predetermined genomic polymorphisms.

As used herein, “consensus sequence” refers to a constructed DNAsequence which identifies SNP and Indel polymorphisms in alleles at alocus. Consensus sequence can be based on either strand of DNA at thelocus and states the nucleotide base of either one of each SNP in thelocus and the nucleotide bases of all Indels in the locus. Thus,although a consensus sequence may not be a copy of an actual DNAsequence, a consensus sequence is useful for precisely designing primersand probes for actual polymorphisms in the locus.

As used herein, the term “single nucleotide polymorphism,” also referredto by the abbreviation “SNP,” means a polymorphism at a single sitewherein said polymorphism constitutes a single base pair change, aninsertion of one or more base pairs, or a deletion of one or more basepairs.

As used herein, the term “haplotype” means a chromosomal region within ahaplotype window defined by an allele of at least one polymorphicmolecular marker. The unique marker fingerprint combinations in eachhaplotype window define individual haplotypes for that window. Further,changes in a haplotype, brought about by recombination for example, mayresult in the modification of a haplotype so that it comprises only aportion of the original (parental) haplotype operably linked to thetrait, for example, via physical linkage to a gene, QTL, or transgene.Any such change in a haplotype would be included in our definition ofwhat constitutes a haplotype so long as the functional integrity of thatgenomic region is unchanged or improved.

As used herein, the term “haplotype window” means a chromosomal regionthat is established by statistical analyses known to those of skill inthe art and is in linkage disequilibrium. Thus, identity by statebetween two inbred individuals (or two gametes) at one or more molecularmarker loci located within this region is taken as evidence ofidentity-by-descent of the entire region. Each haplotype window includesat least one polymorphic molecular marker. Haplotype windows can bemapped along each chromosome in the genome. Haplotype windows are notfixed per se and, given the ever-increasing density of molecularmarkers, this invention anticipates the number and size of haplotypewindows to evolve, with the number of windows increasing and theirrespective sizes decreasing, thus resulting in an ever-increasing degreeconfidence in ascertaining identity by descent based on the identity bystate at the marker loci.

As used herein, “genotype” means the genetic component of the phenotypeand it can be indirectly characterized using markers or directlycharacterized by nucleic acid sequencing. Suitable markers include aphenotypic character, a metabolic profile, a genetic marker, or someother type of marker. A genotype may constitute an allele for at leastone genetic marker locus or a haplotype for at least one haplotypewindow. In some embodiments, a genotype may represent a single locus andin others it may represent a genome-wide set of loci. In anotherembodiment, the genotype can reflect the sequence of a portion of achromosome, an entire chromosome, a portion of the genome, and theentire genome.

As used herein, “phenotype” means the detectable characteristics of acell or organism which can be influenced by gene expression.

As used herein, “linkage” refers to relative frequency at which types ofgametes are produced in a cross. For example, if locus A has genes “A”or “a” and locus B has genes “B” or “b” and a cross between parent Iwith AABB and parent B with aabb will produce four possible gameteswhere the genes are segregated into AB, Ab, aB and ab. The nullexpectation is that there will be independent equal segregation intoeach of the four possible genotypes, i.e. with no linkage ¼ of thegametes will of each genotype. Segregation of gametes into a genotypesdiffering from ¼ are attributed to linkage.

As used herein, “linkage disequilibrium” is defined in the context ofthe relative frequency of gamete types in a population of manyindividuals in a single generation. If the frequency of allele A is p, ais p′, B is q and b is q′, then the expected frequency (with no linkagedisequilibrium) of genotype AB is pq, Ab is pq′, aB is p′q and ab isp′q′. Any deviation from the expected frequency is called linkagedisequilibrium. Two loci are said to be “genetically linked” when theyare in linkage disequilibrium.

As used herein, “quantitative trait locus (QTL)” means a locus thatcontrols to some degree numerically representable traits that areusually continuously distributed.

As used herein, “immunity” means an ASR disease phenotype exhibiting nolesions visible to the unaided eye, or red-brown lesions not associatedwith pustules or viable spores and having a length averaging no largerthan about one fourth the average length of lesions of the susceptiblephenotype assayed under comparable conditions, and covering no more thanabout one twentieth of the leaf surface area. The numerical score forcomplete immunity is 1; for immunity with small but visible lesions ordiscolorations, the numerical score is 1.5.

As used herein, “resistance” means an ASR disease phenotype exhibitingeither immunity or red-brown lesions that may or may not be associatedwith pustules or viable spores, or may be delayed in sporulation, havinga length averaging about one fourth to the same length as the averagelength of lesions of the susceptible phenotype assayed under comparableconditions, the degree of resistance varying inversely with thepercentage of the leaf surface area coverage. The numerical score forresistance is 2 if less than about 50% of the leaf is covered withlesions, and 3 if greater than about 50% of the leaf is covered. A leafhaving about 50% of its area covered with red-brown lesions has anumerical score of 2.5.

As used herein, “susceptibility” means an ASR disease phenotypeexhibiting tan lesions associated with pustules containing viable sporesand having a length averaging about 2 mm to 5 mm under the standardassay conditions used (U.S. Appl 11805667), the degree of susceptibilityvarying directly with the percentage of the leaf surface area coverage.The numerical score for susceptibility is 4 if less than about 50% ofthe leaf is covered with lesions, and 5 if greater than about 50% of theleaf is covered. A leaf having about 50% of its area covered with tanlesions has a numerical score of 4.5.

A response score can also reflect an average score for multiple leavesor tests, such that scores may have numerical values between wholenumbers, typically expressed as decimals.

As used herein, “resistance allele” means the isolated nucleic acidsequence that includes the polymorphic allele associated with resistanceto ASR.

As used herein, the term “soybean” means Glycine max and includes allplant varieties that can be bred with soybean, including wild soybeanspecies.

As used herein, the term “comprising” means “including but not limitedto”.

As used herein, the term “elite line” means any line that has resultedfrom breeding and selection for superior agronomic performance. An eliteplant is any plant from an elite line.

The present invention provides an ASR resistance QTL that maps to aregion on Linkage Group G close to Rpp1; however, the present invention,designated ASR resistance locus 14, is distinct from Rpp1 in having ared-brown phenotype of lesions covering about less than 25% of thesurface area of the leaf, whereas Rpp1 typically confers immunity toASR. The present invention also provides methods and compositions forselecting and introgressing a QTL capable of conferring resistance toASR from a source derived from PI291309C. A QTL that is located at ASRresistance locus 14 is provided.

The present invention provides an ASR resistance QTL that maps to aregion on Linkage Group C2; the present invention, designated ASRresistance locus 15, has a red-brown phenotype of lesions covering aboutless than 25% of the surface area of the leaf. The present inventionfurther provides an ASR resistance QTL that maps to a region on LinkageGroup D2; the present invention, designated ASR resistance locus 16, hasa red-brown phenotype of lesions covering about less than 25% of thesurface area of the leaf. The present invention also provides methodsand compositions for selecting and introgressing QTLs capable ofconferring resistance to ASR from a source derived from PI507009. QTLsthat are located at ASR resistance loci 15 and 16 are provided.

In the present invention, an ASR resistance locus, ASR resistance locus14, is located on Linkage Group G. SNP markers used to monitor theintrogression of ASR resistance locus 14 include those selected from thegroup consisting of NS0119675, NS0095012 and NS0102630. Illustrative ASRresistance locus 14 SNP marker DNA sequence SEQ ID NO: 1 can beamplified using the primers indicated as SEQ ID NO: 9 and 10 anddetected with probes indicated as SEQ ID NO: 25 and 26; SEQ ID NO: 2 canbe amplified using the primers indicated as SEQ ID NO: 11 and 12 anddetected with probes indicated as SEQ ID NO: 27 and 28; SEQ ID NO: 3 canbe amplified using the primers indicated as SEQ ID NO: 13 and 14 anddetected with probes indicated as SEQ ID NO: 29 and 30.

Similarly, the present invention, an ASR resistance locus, ASRresistance locus 15, is located on Linkage Group C2. SNP markers used tomonitor the introgression of ASR resistance locus 15 include thoseselected from the group consisting of NS0093385, NS0118716 andNS0127833. Illustrative ASR resistance locus 15 SNP marker DNA sequenceSEQ ID NO: 4 can be amplified using the primers indicated as SEQ ID NO:15 and 16 and detected with probes indicated as SEQ ID NO: 31 and 32;SEQ ID NO: 5 can be amplified using the primers indicated as SEQ ID NO:17 and 18 and detected with probes indicated as SEQ ID NO: 33 and 34;SEQ ID NO: 6 can be amplified using the primers indicated as SEQ ID NO:19 and 20 and detected with probes indicated as SEQ ID NO: 35 and 36.

In the present invention, an ASR resistance locus, ASR resistance locus16, is located on Linkage Group D2. SNP markers used to monitor theintrogression of ASR resistance locus 16 include those selected from thegroup consisting of NS0113966 and NS0118536. Illustrative ASR resistancelocus 16 SNP marker DNA sequence SEQ ID NO: 7 can be amplified using theprimers indicated as SEQ ID NO: 21 and 22 and detected with probesindicated as SEQ ID NO: 37 and 38; SEQ ID NO: 8 can be amplified usingthe primers indicated as SEQ ID NO: 23 and 24 and detected with probesindicated as SEQ ID NO: 39 and 40.

The present invention also provides a soybean plant comprising a nucleicacid molecule selected from the group consisting of SEQ ID NO: 73through 80 and complements thereof. The present invention also providesa soybean plant comprising a nucleic acid molecule selected from thegroup consisting of SEQ ID NO: 1 though 8, fragments thereof, andcomplements of both. The present invention also provides a soybean plantcomprising a nucleic acid molecule selected from the group consisting ofSEQ ID NO: 9 through 72, fragments thereof, and complements of both.

In one aspect, the soybean plant comprises the 3 nucleic acid moleculesSEQ ID NO: 73 through 75 and complements thereof. In another aspect, thesoybean plant comprises the 3 nucleic acid molecules SEQ ID NO: 1through 3, fragments thereof, and complements of both. In a furtheraspect, the soybean plant comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12nucleic acid molecules selected from the group consisting of SEQ ID NO:9 through 14 and 25 through 30, fragments thereof, and complementsthereof.

In another aspect, the soybean plant comprises the 3 nucleic acidmolecules SEQ ID NO: 76 through 78 and complements thereof. In anotheraspect, the soybean plant comprises the 3 nucleic acid molecules SEQ IDNO: 4 through 6, fragments thereof, and complements of both. In afurther aspect, the soybean plant comprises 2, 3, 4, 5, 6, 7, 8, 9, 10,11 or 12 nucleic acid molecules selected from the group consisting ofSEQ ID NO: 15 through 20 and 31 through 36, fragments thereof, andcomplements thereof.

In another aspect, the soybean plant comprises the 2 nucleic acidmolecules SEQ ID NO: 79 and 80 and complements thereof. In anotheraspect, the soybean plant comprises the 2 nucleic acid molecules SEQ IDNO: 7 and 8, fragments thereof, and complements of both. In a furtheraspect, the soybean plant comprises 2, 3, 4, 5, 6, 7 or 8 nucleic acidmolecules selected from the group consisting of SEQ ID NO: 21 through 24and 37 through 40, fragments thereof, and complements thereof.

In another aspect, the soybean plant comprises the 5 nucleic acidmolecules SEQ ID NO: 76 through 80 and complements thereof. In anotheraspect, the soybean plant comprises the 5 nucleic acid molecules SEQ IDNO: 4 through 8, fragments thereof, and complements of both. In afurther aspect, the soybean plant comprises 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 nucleic acid molecules selectedfrom the group consisting of SEQ ID NO: 15 through 24 and 31 through 40,fragments thereof, and complements thereof.

In another aspect, the soybean plant comprises the 6 nucleic acidmolecules SEQ ID NO: 73 through 78 and complements thereof. In anotheraspect, the soybean plant comprises the 6 nucleic acid molecules SEQ IDNO: 1 through 6, fragments thereof, and complements of both. In afurther aspect, the soybean plant comprises 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 nucleic acidmolecules selected from the group consisting of SEQ ID NO: 9 through 20and 25 through 36, fragments thereof, and complements thereof.

In another aspect, the soybean plant comprises the 5 nucleic acidmolecules SEQ ID NO: 73, 74, 75, 79 and 80 and complements thereof. Inanother aspect, the soybean plant comprises the 5 nucleic acid moleculesSEQ ID NO: 1, 2, 3, 7 and 8, fragments thereof, and complements of both.In a further aspect, the soybean plant comprises 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 nucleic acid moleculesselected from the group consisting of SEQ ID NO: 9 through 14, 21through 30 and 37 through 40, fragments thereof, and complementsthereof.

The present invention also provides a soybean plant comprising an ASRresistance locus 14. Said allele may be homozygous or heterozygous. Thepresent invention also provides a soybean plant comprising an ASRresistance locus 15. Said allele may be homozygous or heterozygous. Thepresent invention also provides a soybean plant comprising an ASRresistance locus 16. Said allele may be homozygous or heterozygous.

The present invention also provides a soybean plant comprising two ormore ASR resistance loci from the group consisting of ASR resistanceloci 14, 15 and 16. Said alleles may be homozygous or heterozygous.

In one embodiment, any single ASR resistance locus 14, 15 or 16, or anycombination of these ASR resistance loci, can be combined with one ormore other ASR resistance loci in a breeding program to produce asoybean plant with at least two ASR resistance loci, as described inU.S. patent application Ser. No. 11/805,667.

As used herein, ASR refers to any ASR variant or isolate. A soybeanplant of the present invention can be resistant to one or more fungicapable of causing or inducing ASR. In one aspect, the present inventionprovides plants resistant or tolerant to ASR as well as methods andcompositions for screening soybean plants for resistance orsusceptibility to ASR, caused by the genus Phakopsora. In a preferredaspect, the present invention provides methods and compositions forscreening soybean plants for resistance or susceptibility to Phakopsorapachyrhizi. In another aspect, the present invention provides plantsresistant to and methods and compositions for screening soybean plantsfor resistance or susceptibility to Phakopsora pachyrhizi strain “MBH1,”originally isolated in southeastern Missouri.

The present invention further provides that the selected plant is fromthe group consisting of members of the genus Glycine, more specificallyfrom the group consisting of Glycine arenaria, Glycine argyrea, Glycinecanescens, Glycine clandestine, Glycine curvata, Glycine cyrtoloba,Glycine falcate, Glycine latifolia, Glycine latrobeana, Glycine max,Glycine microphylla, Glycine pescadrensis, Glycine pindanica, Glycinerubiginosa, Glycine soja, Glycine sp., Glycine stenophita, Glycinetabacina and Glycine tomentella.

Plants of the present invention include a soybean plant that has aresistance level of from 1 to 5, 1 being completely immune, 2 beingresistant to substantially resistant, 3 being mid-resistant to partiallyresistant, 4 being mid-susceptible, and 5 being susceptible, whenassayed for resistance or susceptibility to ASR by any method and ratedas such according to the numerical scale described herein.

In a preferred aspect, the present invention provides a soybean plant tobe assayed for resistance or susceptibility to ASR by any method todetermine whether a soybean plant has a resistance level of from 1 to 5,1 being completely immune, 2 being resistant to substantially resistant,3 being mid-resistant to partially resistant, 4 being mid-susceptible,and 5 being susceptible, according to the numerical scale describedherein.

In light of the generally acknowledged impact of ASR on yield, anotheraspect of the present invention provides plants and derivatives thereofof soybean with one or more ASR resistance loci that exhibit increasedgrain yield in the presence of ASR compared to soybean plants lackingASR resistance loci. In certain embodiments, the increase in grain ofplants of the invention in the presence of ASR may be at least 0.5, 1,1.5, 2.0, 2.5, or 3 bushels/acre as compared to soybean plants lackingASR resistance loci.

A disease resistance QTL of the present invention may be introduced intoan elite soybean inbred line. An “elite line” is any line that hasresulted from breeding and selection for superior agronomic performance.Non-limiting examples of elite soybean varieties that are commerciallyavailable to farmers or soybean breeders include AG00802, A0868, AG0902,A1923, AG2403, A2824, A3704, A4324, A5404, AG5903 and AG6202 (AsgrowSeeds, Des Moines, Iowa, USA); BPR0144RR, BPR 4077NRR and BPR 4390NRR(Bio Plant Research, Camp Point, Ill., USA); DKB17-51 and DKB37-51(DeKalb Genetics, DeKalb, Ill., USA); and DP 4546 RR, and DP 7870 RR(Delta & Pine Land Company, Lubbock, Tex., USA); JG 03R501, JG 32R606CADD and JG 55R503C (JGL Inc., Greencastle, Ind., USA); NKS13-K2 (NKDivision of Syngenta Seeds, Golden Valley, Minn., USA); 90M01, 91M30,92M33, 93M11, 94M30, 95M30 and 97B52 (Pioneer Hi-Bred International,Johnston, Iowa, USA); SG4771NRR and SG5161NRR/STS (Soygenetics, LLC,Lafayette, Ind., USA); 500-K5, S11-L2, S28-Y2, S43-B1, S53-A1, S76-L9and S78-G6 (Syngenta Seeds, Henderson, Ky., USA). An elite plant is arepresentative plant from an elite variety.

An ASR resistance locus of the present invention may also be introducedinto an elite soybean plant comprising one or more transgenes conferringherbicide tolerance, increased yield, insect control, fungal diseaseresistance, virus resistance, nematode resistance, bacterial diseaseresistance, mycoplasma disease resistance, modified oils production,high oil production, high protein production, germination and seedlinggrowth control, enhanced animal and human nutrition, low raffinose,environmental stress resistant, increased digestibility, industrialenzymes, pharmaceutical proteins, peptides and small molecules, improvedprocessing traits, improved flavor, nitrogen fixation, hybrid seedproduction, reduced allergenicity, biopolymers, and biofuels amongothers. In one aspect, the herbicide tolerance is selected from thegroup consisting of glyphosate, dicamba, glufosinate, sulfonylurea,bromoxynil and norflurazon herbicides. These traits can be provided bymethods of plant biotechnology as transgenes in soybean.

A disease resistance QTL allele or alleles can be introduced from anyplant that contains that allele (donor) to any recipient soybean plant.In one aspect, the recipient soybean plant can contain additional ASRresistance loci. In another aspect, the recipient soybean plant cancontain a transgene. In another aspect, while maintaining the introducedQTL, the genetic contribution of the plant providing the diseaseresistance QTL can be reduced by back-crossing or other suitableapproaches. In one aspect, the nuclear genetic material derived from thedonor material in the soybean plant can be less than or about 50%, lessthan or about 25%, less than or about 13%, less than or about 5%, 3%, 2%or 1%, but that genetic material contains the ASR resistance locus ofinterest.

Plants containing one or more ASR resistance loci described can be donorplants. Soybean plants containing resistance loci can be, for example,screened for by using a nucleic acid molecule capable of detecting amarker polymorphism associated with resistance. In one aspect, a donorplant is selected from the group consisting of PI291309C and PI507009.In another aspect, a donor plant is derived from PI291309C or fromPI507009. In another aspect, a donor plant is derived from bothPI291309C and PI507009 by a sexual cross, transformation or other genecombination method that brings at least one ASR resistance gene fromeach of these lines together in the donor plant. A donor plant can be asusceptible line. In one aspect, a donor plant can also be a recipientsoybean plant.

It is further understood that a soybean plant of the present inventionmay exhibit the characteristics of any relative maturity group. In anaspect, the maturity group is selected from the group consisting of 000,00, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10.

An allele of a QTL can, of course, comprise multiple genes or othergenetic factors even within a contiguous genomic region or linkagegroup, such as a haplotype. As used herein, an allele of a diseaseresistance locus can therefore encompass more than one gene or othergenetic factor where each individual gene or genetic component is alsocapable of exhibiting allelic variation and where each gene or geneticfactor is also capable of eliciting a phenotypic effect on thequantitative trait in question. In an aspect of the present inventionthe allele of a QTL comprises one or more genes or other genetic factorsthat are also capable of exhibiting allelic variation. The use of theterm “an allele of a QTL” is thus not intended to exclude a QTL thatcomprises more than one gene or other genetic factor. Specifically, an“allele of a QTL” in the present invention can denote a haplotype withina haplotype window wherein a phenotype can be disease resistance. Ahaplotype window is a contiguous genomic region that can be defined, andtracked, with a set of one or more polymorphic markers wherein thepolymorphisms indicate identity by descent. A haplotype within thatwindow can be defined by the unique fingerprint of alleles at eachmarker. As used herein, an allele is one of several alternative forms ofa gene occupying a given locus on a chromosome. When all the allelespresent at a given locus on a chromosome are the same, that plant ishomozygous at that locus. If the alleles present at a given locus on achromosome differ, that plant is heterozygous at that locus. Plants ofthe present invention may be homozygous or heterozygous at anyparticular ASR locus or for a particular polymorphic marker.

The present invention also provides for parts of the plants of thepresent invention. Plant parts, without limitation, include seed,endosperm, ovule, pollen, stems, cuttings, cells, protoplasts, andtissue cultures. In a particularly preferred aspect of the presentinvention, the plant part is a seed.

The present invention also provides a container of soybean seed in whichgreater than 50%, 60%, 70%, 80%, 90%, 95%, or 99% of the seeds compriseat least one locus from the group consisting of ASR resistance loci 14,15 and 16.

The container of soybean seeds can contain any number, weight, or volumeof seeds. For example, a container can contain at lest, or greater than,about 10, 25, 50, 100, 200, 300, 400, 500, 600, 700, 80, 90, 1000, 1500,2000, 2500, 3000, 3500, 4000 or more seeds. In another aspect, acontainer can contain about, or greater than about, 1 gram, 5 grams, 10grams, 15 grams, 20 grams, 25 grams, 50 grams, 100 grams, 250 grams, 500grams, or 1000 grams of seeds. Alternatively, the container can containat least, or greater than, about 0 ounces, 1 ounce, 5 ounces, 10 ounces,1 pound, 2 pounds, 3 pounds, 4 pounds, 5 pounds, 10 pounds, 15 pounds,20 pounds, 25 pounds, or 50 pounds or more seeds.

Containers of soybean seeds can be any container available in the art.For example, a container can be a box, a bag, a can, a packet, a pouch,a tape roll, a pail, or a tube.

In another aspect, the seeds contained in the containers of soybeanseeds can be treated or untreated soybean seeds. In one aspect, theseeds can be treated to improve germination, for example, by priming theseeds, or by disinfection to protect against seed-born pathogens. Inanother aspect, seeds can be coated with any available coating toimprove, for example, plantability, seed emergence, and protectionagainst seed-born pathogens. Seed coating can be any form of seedcoating including, but not limited to, pelleting, film coating, andencrustments.

Plants or parts thereof of the present invention may be grown in cultureand regenerated. Methods for the regeneration of Glycine max plants fromvarious tissue types and methods for the tissue culture of Glycine maxare known in the art (See, for example, Widholm et al., In VitroSelection and Culture-induced Variation in Soybean, In Soybean:Genetics, Molecular Biology and Biotechnology, Eds. Verma and Shoemaker,CAB International, Wallingford, Oxon, England (1996). Regenerationtechniques for plants such as Glycine max can use as the startingmaterial a variety of tissue or cell types. With Glycine max inparticular, regeneration processes have been developed that begin withcertain differentiated tissue types such as meristems, Cartha et al.,Can. J. Bot. 59:1671-1679 (1981), hypocotyl sections, Cameya et al.,Plant Science Letters 21: 289-294 (1981), and stem node segments, Sakaet al., Plant Science Letters, 19: 193-201 (1980); Cheng et al., PlantScience Letters, 19: 91-99 (1980). Regeneration of whole sexually matureGlycine max plants from somatic embryos generated from explants ofimmature Glycine max embryos has been reported (Ranch et al., In VitroCellular & Developmental Biology 21: 653-658 (1985). Regeneration ofmature Glycine max plants from tissue culture by organogenesis andembryogenesis has also been reported (Barwale et al., Planta 167:473-481 (1986); Wright et al., Plant Cell Reports 5: 150-154 (1986).

The present invention also provides a disease resistant soybean plantselected for by screening for disease resistance or susceptibility inthe soybean plant, the selection comprising interrogating genomicnucleic acids for the presence of a marker molecule that is geneticallylinked to an allele of a QTL associated with disease resistance in thesoybean plant, where the allele of a QTL is also located on a linkagegroup associated with resistance to ASR.

A method of introgressing an allele into a soybean plant comprising (A)crossing at least one first soybean plant comprising a nucleic acidmolecule selected from the group consisting of SEQ ID NO: 73 through 80with at least one second soybean plant in order to form a population,(B) screening the population with one or more nucleic acid markers todetermine if one or more soybean plants from the population contains thenucleic acid molecule, and (C) selecting from the population one or moresoybean plants comprising a nucleic acid molecule selected from thegroup consisting of SEQ ID NO: 73 through 80.

The present invention also includes a method of introgressing an alleleinto a soybean plant comprising: (A) crossing at least one ASR resistantsoybean plant with at least one ASR sensitive soybean plant in order toform a population; (B) screening the population with one or more nucleicacid markers to determine if one or more soybean plants from thepopulation contains at least one ASR resistance allele, wherein each ASRresistance allele is at a resistance locus selected from the groupconsisting of ASR resistance loci 14, 15 and 16.

The present invention includes isolated nucleic acid molecules. Suchmolecules include those nucleic acid molecules capable of detecting apolymorphism genetically or physically linked to an ASR locus. Suchmolecules can be referred to as markers. Additional markers can beobtained that are linked to a locus selected from the group consistingof ASR resistance loci 14, 15 and 16 by available techniques. In oneaspect, the nucleic acid molecule is capable of detecting the presenceor absence of a marker located less than 30, 20, 10, 5, 2, or 1centimorgans from a locus selected from the group consisting of ASRresistance loci 14, 15 and 16. Exemplary nucleic acid molecules withcorresponding map positions are provided in US Patent Application Nos.2005/0204780, 2005/0216545, and Ser. No. 60/932,533, which can be usedto facilitate selection and introgression of the loci of the presentinvention. In another aspect, a marker exhibits a LOD score of 2 orgreater, 3 or greater, or 4 or greater with ASR resistance, measuringusing a method known in the art such as Qgene Version 2.23 (1996) anddefault parameters. In another aspect, the nucleic acid molecule iscapable of detecting a marker in a locus selected from the groupconsisting of the ASR resistance loci 14, 15 and 16. In a furtheraspect, a nucleic acid molecule is selected from the group consisting ofSEQ ID NO: 1 through SEQ ID NO: 80, fragments thereof, complementsthereof, and nucleic acid molecules capable of specifically hybridizingto one or more of these nucleic acid molecules.

In a preferred aspect, a nucleic acid molecule of the present inventionincludes those that will specifically hybridize to one or more of thenucleic acid molecules set forth in SEQ ID NO:1 through SEQ ID NO: 80 orcomplements thereof or fragments of either under moderately stringentconditions, for example at about 2.0×SSC and about 65° C. In aparticularly preferred aspect, a nucleic acid of the present inventionwill specifically hybridize to one or more of the nucleic acid moleculesset forth in SEQ ID NO: 1 through SEQ ID NO: 80 or complements orfragments of either under high stringency conditions. In one aspect ofthe present invention, a preferred marker nucleic acid molecule of thepresent invention has the nucleic acid sequence set forth in SEQ ID NO:1 through SEQ ID NO: 80 or complements thereof or fragments of either.In another aspect of the present invention, a preferred marker nucleicacid molecule of the present invention shares between 80% and 100% or90% and 100% sequence identity with the nucleic acid sequences set forthin SEQ ID NO: 1 through SEQ ID NO: 80 or complements thereof orfragments of either. In a further aspect of the present invention, apreferred marker nucleic acid molecule of the present invention sharesbetween 95% and 100% sequence identity with the sequences set forth inSEQ ID NO: 1 through SEQ ID NO: 80 or complements thereof or fragmentsof either. In a more preferred aspect of the present invention, apreferred marker nucleic acid molecule of the present invention sharesbetween 98% and 100% sequence identity with the nucleic acid sequenceset forth in SEQ ID NO: 1 through SEQ ID NO: 80 or complement thereof orfragments of either. In a more preferred aspect of the presentinvention, a preferred marker nucleic acid molecule of the presentinvention shares between 99% and 100% sequence identity with the nucleicacid sequence set forth in SEQ ID NO: 1 through SEQ ID NO: 80 orcomplement thereof or fragments of either. In a more preferred aspect ofthe present invention, a preferred marker nucleic acid molecule of thepresent invention shares 100% sequence identity with the nucleic acidsequence set forth in SEQ ID NO: 1 through SEQ ID NO: 80 or complementthereof or fragments of either.

Nucleic acid molecules or fragments thereof are capable of specificallyhybridizing to other nucleic acid molecules under certain circumstances.As used herein, two nucleic acid molecules are capable of specificallyhybridizing to one another if the two molecules are capable of formingan anti-parallel, double-stranded nucleic acid structure. A nucleic acidmolecule is the “complement” of another nucleic acid molecule if theyexhibit complete complementarity. As used herein, molecules are exhibit“complete complementarity” when every nucleotide of one of the moleculesis complementary to a nucleotide of the other. Two molecules are“minimally complementary” if they can hybridize to one another withsufficient stability to permit them to remain annealed to one anotherunder at least conventional “low-stringency” conditions. Similarly, themolecules are “complementary” if they can hybridize to one another withsufficient stability to permit them to remain annealed to one anotherunder conventional “high-stringency” conditions. Conventional stringencyconditions are described by Sambrook et al., In: Molecular Cloning, ALaboratory Manual, 2 nd Edition, Cold Spring Harbor Press, Cold SpringHarbor, N.Y. (1989), and by Haymes et al., In: Nucleic AcidHybridization, A Practical Approach, IRL Press, Washington, D.C. (1985).Departures from complete complementarity are therefore permissible, aslong as such departures do not completely preclude the capacity of themolecules to form a double-stranded structure. In order for a nucleicacid molecule to serve as a primer or probe it need only be sufficientlycomplementary in sequence to be able to form a stable double-strandedstructure under the particular solvent and salt concentrations employed.

As used herein, a substantially homologous sequence is a nucleic acidsequence that will specifically hybridize to the complement of thenucleic acid sequence to which it is being compared under highstringency conditions. The nucleic-acid probes and primers of thepresent invention can hybridize under stringent conditions to a targetDNA sequence. The term “stringent hybridization conditions” is definedas conditions under which a probe or primer hybridizes specifically witha target sequence(s) and not with non-target sequences, as can bedetermined empirically. The term “stringent conditions” is functionallydefined with regard to the hybridization of a nucleic-acid probe to atarget nucleic acid (i.e., to a particular nucleic-acid sequence ofinterest) by the specific hybridization procedure discussed in Sambrooket al., 1989, at 9.52-9.55. See also, Sambrook et al., 1989 at9.47-9.52, 9.56-9.58; Kanehisa 1984 Nucl. Acids Res. 12:203-213; andWetmur et al. 1968 J. Mol. Biol. 31:349-370. Appropriate stringencyconditions that promote DNA hybridization are, for example, 6.0× sodiumchloride/sodium citrate (SSC) at about 45° C., followed by a wash of2.0×SSC at 50° C., are known to those skilled in the art or can be foundin Current Protocols in Molecular Biology, John Wiley & Sons, N.Y.,1989, 6.3.1-6.3.6. For example, the salt concentration in the wash stepcan be selected from a low stringency of about 2.0×SSC at 50° C. to ahigh stringency of about 0.2×SSC at 50° C. In addition, the temperaturein the wash step can be increased from low stringency conditions at roomtemperature, about 22° C., to high stringency conditions at about 65° C.Both temperature and salt may be varied, or either the temperature orthe salt concentration may be held constant while the other variable ischanged.

For example, hybridization using DNA or RNA probes or primers can beperformed at 65° C. in 6×SSC, 0.5% SDS, 5×Denhardt's, 100 μg/mLnonspecific DNA (e.g., sonicated salmon sperm DNA) with washing at0.5×SSC, 0.5% SDS at 65° C., for high stringency.

It is contemplated that lower stringency hybridization conditions suchas lower hybridization and/or washing temperatures can be used toidentify related sequences having a lower degree of sequence similarityif specificity of binding of the probe or primer to target sequence(s)is preserved. Accordingly, the nucleotide sequences of the presentinvention can be used for their ability to selectively form duplexmolecules with complementary stretches of DNA, RNA, or cDNA fragments.

A fragment of a nucleic acid molecule can be any sized fragment andillustrative fragments include fragments of nucleic acid sequences setforth in SEQ ID NO: 1 through SEQ ID NO: 80 and complements thereof. Inone aspect, a fragment can be between 15 and 25, 15 and 30, 15 and 40,15 and 50, 15 and 100, 20 and 25, 20 and 30, 20 and 40, 20 and 50, and100, 25 and 30, 25 and 40, 25 and 50, 25 and 100, 30 and 40, 30 and 50,and 30 and 100 nucleotides. In another aspect, the fragment can begreater than 10, 15, 20, 25, 30, 35, 40, 50, 100, or 250 nucleotides.

Additional genetic markers can be used to select plants with an alleleof a QTL associated with fungal disease resistance of soybeans of thepresent invention. Examples of public marker databases include, forexample Soybase, an Agricultural Research Service, United StatesDepartment of Agriculture.

Genetic markers of the present invention include “dominant” or“codominant” markers. “Codominant markers” reveal the presence of two ormore alleles (two per diploid individual). “Dominant markers” reveal thepresence of only a single allele. The presence of the dominant markerphenotype (e.g., a band of DNA) is an indication that one allele ispresent in either the homozygous or heterozygous condition. The absenceof the dominant marker phenotype (e.g., absence of a DNA band) is merelyevidence that “some other” undefined allele is present. In the case ofpopulations where individuals are predominantly homozygous and loci arepredominantly dimorphic, dominant and codominant markers can be equallyvaluable. As populations become more heterozygous and multiallelic,codominant markers often become more informative of the genotype thandominant markers.

In another embodiment, markers, such as single sequence repeat markers(SSR), AFLP markers, RFLP markers, RAPD markers, phenotypic markers,isozyme markers, single nucleotide polymorphisms (SNPs), insertions ordeletions (Indels), single feature polymorphisms (SFPs, for example, asdescribed in Borevitz et al. 2003 Gen. Res. 13:513-523), microarraytranscription profiles, DNA-derived sequences, and RNA-derived sequencesthat are genetically linked to or correlated with alleles of a QTL ofthe present invention can be utilized.

In one embodiment, nucleic acid-based analyses for the presence orabsence of the genetic polymorphism can be used for the selection ofseeds in a breeding population. A wide variety of genetic markers forthe analysis of genetic polymorphisms are available and known to thoseof skill in the art. The analysis may be used to select for genes, QTL,alleles, or genomic regions (haplotypes) that comprise or are linked toa genetic marker.

Herein, nucleic acid analysis methods are known in the art and include,but are not limited to, PCR-based detection methods (for example, TaqManassays), microarray methods, and nucleic acid sequencing methods. In oneembodiment, the detection of polymorphic sites in a sample of DNA, RNA,or cDNA may be facilitated through the use of nucleic acid amplificationmethods. Such methods specifically increase the concentration ofpolynucleotides that span the polymorphic site, or include that site andsequences located either distal or proximal to it. Such amplifiedmolecules can be readily detected by gel electrophoresis, fluorescencedetection methods, or other means.

A method of achieving such amplification employs the polymerase chainreaction (PCR) (Mullis et al. 1986 Cold Spring Harbor Symp. Quant. Biol.51:263-273; European Patent 50,424; European Patent 84,796; EuropeanPatent 258,017; European Patent 237,362; European Patent 201,184; U.S.Pat. No. 4,683,202; U.S. Pat. No. 4,582,788; and U.S. Pat. No.4,683,194), using primer pairs that are capable of hybridizing to theproximal sequences that define a polymorphism in its double-strandedform

Polymorphisms in DNA sequences can be detected or typed by a variety ofeffective methods well known in the art including, but not limited to,those disclosed in U.S. Pat. Nos. 5,468,613 and 5,217,863; 5,210,015;5,876,930; 6,030,787; 6,004,744; 6,013,431; 5,595,890; 5,762,876;5,945,283; 5,468,613; 6,090,558; 5,800,944; and 5,616,464, all of whichare incorporated herein by reference in their entireties. However, thecompositions and methods of this invention can be used in conjunctionwith any polymorphism typing method to type polymorphisms in soybeangenomic DNA samples. These soybean genomic DNA samples used include butare not limited to soybean genomic DNA isolated directly from a soybeanplant, cloned soybean genomic DNA, or amplified soybean genomic DNA.

For instance, polymorphisms in DNA sequences can be detected byhybridization to allele-specific oligonucleotide (ASO) probes asdisclosed in U.S. Pat. Nos. 5,468,613 and 5,217,863. U.S. Pat. No.5,468,613 discloses allele specific oligonucleotide hybridizations wheresingle or multiple nucleotide variations in nucleic acid sequence can bedetected in nucleic acids by a process in which the sequence containingthe nucleotide variation is amplified, spotted on a membrane and treatedwith a labeled sequence-specific oligonucleotide probe.

Target nucleic acid sequence can also be detected by probe ligationmethods as disclosed in U.S. Pat. No. 5,800,944 where sequence ofinterest is amplified and hybridized to probes followed by ligation todetect a labeled part of the probe.

Microarrays can also be used for polymorphism detection, whereinoligonucleotide probe sets are assembled in an overlapping fashion torepresent a single sequence such that a difference in the targetsequence at one point would result in partial probe hybridization(Borevitz et al., Genome Res. 13:513-523 (2003); Cui et al.,Bioinformatics 21:3852-3858 (2005). On any one microarray, it isexpected there will be a plurality of target sequences, which mayrepresent genes and/or noncoding regions wherein each target sequence isrepresented by a series of overlapping oligonucleotides, rather than bya single probe. This platform provides for high throughput screening aplurality of polymorphisms. A single-feature polymorphism (SFP) is apolymorphism detected by a single probe in an oligonucleotide array,wherein a feature is a probe in the array. Typing of target sequences bymicroarray-based methods is disclosed in U.S. Pat. Nos. 6,799,122;6,913,879; and 6,996,476.

Target nucleic acid sequence can also be detected by probe linkingmethods as disclosed in U.S. Pat. No. 5,616,464 employing at least onepair of probes having sequences homologous to adjacent portions of thetarget nucleic acid sequence and having side chains which non-covalentlybind to form a stem upon base pairing of said probes to said targetnucleic acid sequence. At least one of the side chains has aphotoactivatable group which can form a covalent cross-link with theother side chain member of the stem.

Other methods for detecting SNPs and Indels include single baseextension (SBE) methods. Examples of SBE methods include, but are notlimited, to those disclosed in U.S. Pat. Nos. 6,004,744; 6,013,431;5,595,890; 5,762,876; and 5,945,283. SBE methods are based on extensionof a nucleotide primer that is adjacent to a polymorphism to incorporatea detectable nucleotide residue upon extension of the primer. In certainembodiments, the SBE method uses three synthetic oligonucleotides. Twoof the oligonucleotides serve as PCR primers and are complementary tosequence of the locus of soybean genomic DNA which flanks a regioncontaining the polymorphism to be assayed. Following amplification ofthe region of the soybean genome containing the polymorphism, the PCRproduct is mixed with the third oligonucleotide (called an extensionprimer) which is designed to hybridize to the amplified DNA adjacent tothe polymorphism in the presence of DNA polymerase and twodifferentially labeled dideoxynucleosidetriphosphates. If thepolymorphism is present on the template, one of the labeleddideoxynucleosidetriphosphates can be added to the primer in a singlebase chain extension. The allele present is then inferred by determiningwhich of the two differential labels was added to the extension primer.Homozygous samples will result in only one of the two labeled basesbeing incorporated and thus only one of the two labels will be detected.Heterozygous samples have both alleles present, and will thus directincorporation of both labels (into different molecules of the extensionprimer) and thus both labels will be detected.

In a preferred method for detecting polymorphisms, SNPs and Indels canbe detected by methods disclosed in U.S. Pat. Nos. 5,210,015; 5,876,930;and 6,030,787 in which an oligonucleotide probe having a 5′ fluorescentreporter dye and a 3′quencher dye covalently linked to the 5′ and 3′ends of the probe. When the probe is intact, the proximity of thereporter dye to the quencher dye results in the suppression of thereporter dye fluorescence, e.g. by Forster-type energy transfer. DuringPCR forward and reverse primers hybridize to a specific sequence of thetarget DNA flanking a polymorphism while the hybridization probehybridizes to polymorphism-containing sequence within the amplified PCRproduct. In the subsequent PCR cycle DNA polymerase with 5′→3′exonuclease activity cleaves the probe and separates the reporter dyefrom the quencher dye resulting in increased fluorescence of thereporter.

For the purpose of QTL mapping, the markers included should bediagnostic of origin in order for inferences to be made about subsequentpopulations. SNP markers are ideal for mapping because the likelihoodthat a particular SNP allele is derived from independent origins in theextant populations of a particular species is very low. As such, SNPmarkers are useful for tracking and assisting introgression of QTLs,particularly in the case of haplotypes.

The genetic linkage of additional marker molecules can be established bya gene mapping model such as, without limitation, the flanking markermodel reported by Lander et al. (Lander et al. 1989 Genetics,121:185-199), and the interval mapping, based on maximum likelihoodmethods described therein, and implemented in the software packageMAPMAKER/QTL (Lincoln and Lander, Mapping Genes Controlling QuantitativeTraits Using MAPMAKER/QTL, Whitehead Institute for Biomedical Research,Massachusetts, (1990). Additional software includes Qgene, Version 2.23(1996), Department of Plant Breeding and Biometry, 266 Emerson Hall,Cornell University, Ithaca, N.Y.). Use of Qgene software is aparticularly preferred approach.

A maximum likelihood estimate (MLE) for the presence of a marker iscalculated, together with an MLE assuming no QTL effect, to avoid falsepositives. A log₁₀ of an odds ratio (LOD) is then calculated as:LOD=log₁₀ (MLE for the presence of a QTL/MLE given no linked QTL). TheLOD score essentially indicates how much more likely the data are tohave arisen assuming the presence of a QTL versus in its absence. TheLOD threshold value for avoiding a false positive with a givenconfidence, say 95%, depends on the number of markers and the length ofthe genome. Graphs indicating LOD thresholds are set forth in Lander etal. (1989), and further described by Arús and Moreno-Gonzalez, PlantBreeding, Hayward, Bosemark, Romagosa (eds.) Chapman & Hall, London, pp.314-331 (1993).

Additional models can be used. Many modifications and alternativeapproaches to interval mapping have been reported, including the use ofnon-parametric methods (Kruglyak et al. 1995 Genetics, 139:1421-1428).Multiple regression methods or models can be also be used, in which thetrait is regressed on a large number of markers (Jansen, Biometrics inPlant Breed, van Oijen, Jansen (eds.) Proceedings of the Ninth Meetingof the Eucarpia Section Biometrics in Plant Breeding, The Netherlands,pp. 116-124 (1994); Weber and Wricke, Advances in Plant Breeding,Blackwell, Berlin, 16 (1994)). Procedures combining interval mappingwith regression analysis, whereby the phenotype is regressed onto asingle putative QTL at a given marker interval, and at the same timeonto a number of markers that serve as ‘cofactors,’ have been reportedby Jansen et al. (Jansen et al. 1994 Genetics, 136:1447-1455) and Zeng(Zeng 1994 Genetics 136:1457-1468). Generally, the use of cofactorsreduces the bias and sampling error of the estimated QTL positions (Utzand Melchinger, Biometrics in Plant Breeding, van Oijen, Jansen (eds.)Proceedings of the Ninth Meeting of the Eucarpia Section Biometrics inPlant Breeding, The Netherlands, pp. 195-204 (1994), thereby improvingthe precision and efficiency of QTL mapping (Zeng 1994). These modelscan be extended to multi-environment experiments to analyzegenotype-environment interactions (Jansen et al. 1995 Theor. Appl.Genet. 91:33-3).

Selection of appropriate mapping populations is important to mapconstruction. The choice of an appropriate mapping population depends onthe type of marker systems employed (Tanksley et al., Molecular mappingin plant chromosomes. chromosome structure and function: Impact of newconcepts J. P. Gustafson and R. Appels (Eds.) Plenum Press, New York,pp. 157-173 (1988)). Consideration must be given to the source ofparents (adapted vs. exotic) used in the mapping population. Chromosomepairing and recombination rates can be severely disturbed (suppressed)in wide crosses (adapted×exotic) and generally yield greatly reducedlinkage distances. Wide crosses will usually provide segregatingpopulations with a relatively large array of polymorphisms when comparedto progeny in a narrow cross (adapted×adapted).

An F₂ population is the first generation of self pollinating (selfing).Usually a single F₁ plant is selfed to generate a population segregatingfor all the genes in Mendelian (1:2:1) fashion. Maximum geneticinformation is obtained from a completely classified F₂ population usinga codominant marker system (Mather, Measurement of Linkage in Heredity:Methuen and Co., (1938)). In the case of dominant markers, progeny tests(e.g. F₃, BCF₂) are required to identify the heterozygotes, thus makingit equivalent to a completely classified F₂ population. However, thisprocedure is often prohibitive because of the cost and time involved inprogeny testing. Progeny testing of F₂ individuals is often used in mapconstruction where phenotypes do not consistently reflect genotype (e.g.disease resistance) or where trait expression is controlled by a QTL.Segregation data from progeny test populations (e.g. F₃ or BCF₂) can beused in map construction. Marker-assisted selection can then be appliedto cross progeny based on marker-trait map associations (F₂, F₃), wherelinkage groups have not been completely disassociated by recombinationevents (i.e., maximum disequilibrium).

Recombinant inbred lines (RIL) (genetically related lines; usually >F₅,developed from continuously selfing F₂ lines towards homozygosity) canbe used as a mapping population. Information obtained from dominantmarkers can be maximized by using RIL because all loci are homozygous ornearly so. Under conditions of tight linkage (i.e., about <10%recombination), dominant and co-dominant markers evaluated in RILpopulations provide more information per individual than either markertype in backcross populations (Reiter et al. 1992 Proc. Natl. Acad. Sci.(USA) 89:1477-1481). However, as the distance between markers becomeslarger (i.e., loci become more independent), the information in RILpopulations decreases dramatically.

Backcross populations (e.g., generated from a cross between a successfulvariety (recurrent parent) and another variety (donor parent) carrying atrait not present in the former) can be utilized as a mappingpopulation. A series of backcrosses to the recurrent parent can be madeto recover most of its desirable traits. Thus a population is createdconsisting of individuals nearly like the recurrent parent but eachindividual carries varying amounts or mosaic of genomic regions from thedonor parent. Backcross populations can be useful for mapping dominantmarkers if all loci in the recurrent parent are homozygous and the donorand recurrent parent have contrasting polymorphic marker alleles (Reiteret al. 1992).

Information obtained from backcross populations using either codominantor dominant markers is less than that obtained from F₂ populationsbecause one, rather than two, recombinant gametes are sampled per plant.Backcross populations, however, are more informative (at low markersaturation) when compared to RILs as the distance between linked lociincreases in RIL populations (i.e. about 0.15% recombination). Increasedrecombination can be beneficial for resolution of tight linkages, butmay be undesirable in the construction of maps with low markersaturation.

Near-isogenic lines (NIL) created by many backcrosses to produce anarray of individuals that are nearly identical in genetic compositionexcept for the trait or genomic region under interrogation can be usedas a mapping population. In mapping with NILs, only a portion of thepolymorphic loci are expected to map to a selected region.

Bulk segregant analysis (BSA) is a method developed for the rapididentification of linkage between markers and traits of interest(Michelmore et al. 1991 Proc. Natl. Acad. Sci. (U.S.A.) 88:9828-9832).In BSA, two bulked DNA samples are drawn from a segregating populationoriginating from a single cross. These bulks contain individuals thatare identical for a particular trait (resistant or susceptible toparticular disease) or genomic region but arbitrary at unlinked regions(i.e. heterozygous). Regions unlinked to the target region will notdiffer between the bulked samples of many individuals in BSA.

Plants of the present invention can be part of or generated from abreeding program. The choice of breeding method depends on the mode ofplant reproduction, the heritability of the trait(s) being improved, andthe type of cultivar used commercially (e.g., F₁ hybrid cultivar,pureline cultivar, etc). A cultivar is a race or variety of a plantspecies that has been created or selected intentionally and maintainedthrough cultivation.

As used herein, the term progeny refers to a genetic descendant. Thepresent invention provides for progeny produced by sexual or vegetativereproduction, grown from seeds, regenerated from the above-describedplant parts, or regenerated from tissue culture of a cultivar or aprogeny plant.

Molecular breeding is often referred to as marker-assisted selection(MAS) and marker-assisted breeding (MAB), wherein MAS refers to makingbreeding decisions on the basis of molecular marker genotypes and MAB isa general term representing the use of molecular markers in plantbreeding. In these types of molecular breeding programs, genetic markeralleles can be used to identify plants that contain the desired genotypeat one marker locus, several loci, or a haplotype, and that would beexpected to transfer the desired genotype, along with a desiredphenotype to their progeny. Markers are highly useful in plant breedingbecause once established, they are not subject to environmental orepistatic interactions. Furthermore, certain types of markers are suitedfor high throughput detection, enabling rapid identification in a costeffective manner.

Selected, non-limiting approaches for breeding the plants of the presentinvention are set forth below. A breeding program can be enhanced usingmarker assisted selection (MAS) on the progeny of any cross. MAS is aselection process where a trait of interest is selected not based on thetrait itself but on a marker linked to it. For example if MAS is beingused to select individuals with a disease, the level of disease is notquantified but rather a marker allele which is linked with disease isused to determine disease presence. The assumption is that linked alleleassociates with the gene and/or quantitative trait locus (QTL) ofinterest. MAS can be useful for traits that are difficult to measure,expensive to phenotype, exhibit low heritability, and/or are expressedlate in plant development. It is understood that nucleic acid markers ofthe present invention can be used in a MAS (breeding) program. It isfurther understood that any commercial and non-commercial cultivars canbe utilized in a breeding program. Factors such as, for example,emergence vigor, vegetative vigor, stress tolerance, disease resistance,branching, flowering, seed set, seed size, seed density, standability,and threshability etc. will generally dictate the choice.

For highly heritable traits, a choice of superior individual plantsevaluated at a single location will be effective, whereas for traitswith low heritability, selection should be based on mean values obtainedfrom replicated evaluations of families of related plants. Popularselection methods commonly include pedigree selection, modified pedigreeselection, mass selection, and recurrent selection. In a preferredaspect, a backcross or recurrent breeding program is undertaken.

The complexity of inheritance influences choice of the breeding method.Backcross breeding can be used to transfer one or a few favorable genesfor a highly heritable trait into a desirable cultivar. This approachhas been used extensively for breeding disease-resistant cultivars.Various recurrent selection techniques are used to improvequantitatively inherited traits controlled by numerous genes.

Breeding lines can be tested and compared to appropriate standards inenvironments representative of the commercial target area(s) for two ormore generations. The best lines are candidates for new commercialcultivars; those still deficient in traits may be used as parents toproduce new populations for further selection.

Pedigree breeding and recurrent selection breeding methods can be usedto develop cultivars from breeding populations. Breeding programscombine desirable traits from two or more cultivars or variousbroad-based sources into breeding pools from which cultivars aredeveloped by selfing and selection of desired phenotypes. New cultivarscan be evaluated to determine which have commercial potential.

Backcross breeding has been used to transfer genes for a simplyinherited, highly heritable trait into a desirable homozygous cultivaror inbred line, which is the recurrent parent. The source of the traitto be transferred is called the donor parent. After the initial cross,individuals possessing the phenotype of the donor parent are selectedand repeatedly crossed (backcrossed) to the recurrent parent. Theresulting plant is expected to have most attributes of the recurrentparent (e.g., cultivar) and, in addition, the desirable traittransferred from the donor parent.

The single-seed descent procedure in the strict sense refers to plantinga segregating population, harvesting a sample of one seed per plant, andusing the one-seed sample to plant the next generation. When thepopulation has been advanced from the F₂ to the desired level ofinbreeding, the plants from which lines are derived will each trace todifferent F₂ individuals. The number of plants in a population declineseach generation due to failure of some seeds to germinate or some plantsto produce at least one seed. As a result, not all of the F₂ plantsoriginally sampled in the population will be represented by a progenywhen generation advance is completed.

Descriptions of other breeding methods that are commonly used fordifferent traits and crops can be found in one of several referencebooks (Allard, “Principles of Plant Breeding,” John Wiley & Sons, NY, U.of CA, Davis, Calif., 50-98, 1960; Simmonds, “Principles of cropimprovement,” Longman, Inc., NY, 369-399, 1979; Sneep and Hendriksen,“Plant breeding perspectives,” Wageningen (ed), Center for AgriculturalPublishing and Documentation, 1979; Fehr, In: Soybeans: Improvement,Production and Uses, 2nd Edition, Monograph., 16:249, 1987; Fehr,“Principles of variety development,” Theory and Technique, (Vol. 1) andCrop Species Soybean (Vol. 2), Iowa State Univ., Macmillan Pub. Co., NY,360-376, 1987).

An alternative to traditional QTL mapping involves achieving higherresolution by mapping haplotypes, versus individual markers (Fan et al.2006 Genetics 172:663-686). This approach tracks blocks of DNA known ashaplotypes, as defined by polymorphic markers, which are assumed to beidentical by descent in the mapping population. This assumption resultsin a larger effective sample size, offering greater resolution of QTL.Methods for determining the statistical significance of a correlationbetween a phenotype and a genotype, in this case a haplotype, may bedetermined by any statistical test known in the art and with anyaccepted threshold of statistical significance being required. Theapplication of particular methods and thresholds of significance arewell with in the skill of the ordinary practitioner of the art.

It is further understood, that the present invention provides bacterial,viral, microbial, insect, mammalian and plant cells comprising thenucleic acid molecules of the present invention.

As used herein, a “nucleic acid molecule,” be it a naturally occurringmolecule or otherwise may be “substantially purified,” if desired,referring to a molecule separated from substantially all other moleculesnormally associated with it in its native state. More preferably asubstantially purified molecule is the predominant species present in apreparation. A substantially purified molecule may be greater than 60%free, preferably 75% free, more preferably 90% free, and most preferably95% free from the other molecules (exclusive of solvent) present in thenatural mixture. The term “substantially purified” is not intended toencompass molecules present in their native state.

The agents of the present invention will preferably be “biologicallyactive” with respect to either a structural attribute, such as thecapacity of a nucleic acid to hybridize to another nucleic acidmolecule, or the ability of a protein to be bound by an antibody (or tocompete with another molecule for such binding). Alternatively, such anattribute may be catalytic, and thus involve the capacity of the agentto mediate a chemical reaction or response.

The agents of the present invention may also be recombinant. As usedherein, the term recombinant means any agent (e.g. DNA, peptide etc.),that is, or results, however indirect, from human manipulation of anucleic acid molecule.

The agents of the present invention may be labeled with reagents thatfacilitate detection of the agent (e.g. fluorescent labels (Prober etal. 1987 Science 238:336-340; Albarella et al., European Patent 144914),chemical labels (Sheldon et al., U.S. Pat. No. 4,582,789; Albarella etal., U.S. Pat. No. 4,563,417), modified bases (Miyoshi et al., EuropeanPatent 119448).

Having now generally described the invention, the same will be morereadily understood through reference to the following examples which areprovided by way of illustration, and are not intended to be limiting ofthe present invention, unless specified.

EXAMPLES Example 1 Testing of Soybean Accessions for ASR ResistanceUsing the Detached Leaf Assay

Forty putative ASR resistant accessions were screened for ASRresistance. Leaf assays for resistance to ASR were performed on these 40lines, and appropriate susceptible accessions as controls, as describedin U.S. patent application Ser. No. 11/805,667. Plants were scored ashaving a degree of resistance indicated by a numerical rating from 1 to5; 1—being immune, 2—demonstrating red/brown lesions over less thanabout 50% of the leaf area, 3—demonstrating red/brown lesions overgreater than about 50% of the leaf area, 4—demonstrating tan lesionsover less than about 50% of the leaf area and 5—demonstrating tanlesions over greater than about 50% of the leaf area, i.e. completelysusceptible. An average rust severity score over multiple tests of 1.5was obtained for accession PI291309C, a maturity-group-2 line, and forPI507009, a maturity-group-6 line, when infected with a North Americanisolate of P. pachyrhizi MBH1 from southeastern Missouri.

PI291309C and PI507009 were each crossed with soybean line MV0079 togenerate F2 mapping populations, individuals of which were genotypedwith 104 SNPs and tested for ASR resistance. SNPs were selected based onthe fingerprint profile of the parents and genome coverage. The ASRresistance locus discovered in PI291309C, designated ASR resistancelocus 14, was mapped to linkage group G of the public soybean geneticlinkage map close to the Rpp1 gene (FIG. 1). The ASR resistance locuscharacterized in PI291309C is distinct on a haplotype basis from soybeanlines containing the ASR resistance locus designated Rpp1, as well asdiffering in phenotypic response to southeastern Missouri P. pachyrhizistrain MBH1. Three SNP markers, NS0095012 (P<0.0050) and NS0102630 andNS0119675 (P<0.0010 for each) were found to be in high linkagedisequilibria with the ASR resistance locus 14 disease phenotypicresponse, and therefore associated with ASR resistance locus 14. Allthree SNP markers were identified as being useful in monitoring thepositive introgression of ASR resistance locus 14. SNP marker NS0095012corresponds to SEQ ID NO: 1; NS0119675 corresponds to SEQ ID NO: 2; andNS0102630 corresponds to SEQ ID NO: 3.

The ASR resistance locus 15 was discovered in PI507009 and mapped tolinkage group C2 of the public soybean genetic linkage map (FIG. 2).Three SNP markers, NS0127833 (R Sq>0.050) and NS0118716 and NS0093385 (RSq>0.200 for each) were found to be in high linkage disequilibria withthe ASR resistance locus 15 disease phenotypic response, and thereforeassociated with ASR resistance locus 15. All three SNP markers wereidentified as being useful in monitoring the positive introgression ofASR resistance locus 15. SNP marker NS0093385 corresponds to SEQ ID NO:4; NS0118716 corresponds to SEQ ID NO: 5; and NS0127833 corresponds toSEQ ID NO: 6.

The ASR resistance locus 16 discovered in PI507009 and mapped to linkagegroup D2 of the public soybean genetic linkage map (FIG. 3). Two SNPmarkers, NS0118536 (R Sq>0.050) and NS0113966 (R Sq>0.150) were found tobe in linkage disequilibria with the ASR resistance locus 16 diseasephenotypic response, and therefore associated with ASR resistance locus16. Both SNP markers were identified as being useful in monitoring theintrogression of ASR resistance locus 16. SNP marker NS0113966corresponds to SEQ ID NO: 7; and NS0118536 corresponds to SEQ ID NO: 8.Table 1 lists the SNP markers, their chromosome positions, SNPpositions, the resistance allele and the SEQ ID number for theresistance allele for each SNP.

Example 2 Exemplary Marker Assays for Detecting ASR Resistance

In one embodiment, the detection of polymorphic sites in a sample ofDNA, RNA, or cDNA may be facilitated through the use of nucleic acidamplification methods. Such methods specifically increase theconcentration of polynucleotides that span the polymorphic site, orinclude that site and sequences located either distal or proximal to it.Such amplified molecules can be readily detected by gel electrophoresis,fluorescence detection methods, or other means. Exemplary primers andprobes for amplifying and detecting genomic regions associated with ASRresistance are given in Table 2.

Example 3 Oligonucleotide Hybridization Probes Useful for DetectingSoybean Plants with ASR Resistance Loci

Oligonucleotides can also be used to detect or type the polymorphismsassociated with the ASR resistance locus disclosed herein usinghybridization-based SNP detection methods. Exemplary oligonucleotidescapable of hybridizing to isolated nucleic acid sequences which includethe polymorphism are provided. It is within the skill of the art todesign assays with experimentally determined stringency to discriminatebetween the allelic states of the polymorphisms presented herein.Exemplary assays include Southern blots, Northern blots, microarrays, insitu hybridization, and other methods of polymorphism detection based onhybridization. Exemplary oligonucleotides for use in hybridization-basedSNP detection are provided in Table 3. These oligonucleotides can bedetectably labeled with radioactive labels, fluorophores, or otherchemiluminescent means to facilitate detection of hybridization tosamples of genomic or amplified nucleic acids derived from one or moresoybean plants using methods known in the art.

TABLE 1 Listing of SNP markers for ASR resistance loci 14, 15 and 16with the resistance and susceptibility allele for each marker indicated.ASR SEQ ID NO. Linkage Chromosome resistance SNP Favorable ResistanceSusceptibility of resistance Marker Group Position locus SEQ ID NO.position parent allele allele allele NS0095012 G 85.2 14 1 99 PI291309CG T 73 NS0119675 G 113.0 14 2 53 PI291309C A T 74 NS0102630 G 132.1 14 3186 PI291309C C A 75 NS0093385 C2 129.4 15 4 109 PI507009 T C 76NS0118716 C2 149.9 15 5 366 PI507009 C T 77 NS0127833 C2 171.2 15 6 57PI507009 T C 78 NS0113966 D2 49.6 16 7 332 PI507009 G A 79 NS0118536 D275.5 16 8 286 PI507009 C T 80 The resistance allele corresponds to the35 base pair nucleic acid sequence that includes the polymorphismassociated with ASR resistance.

TABLE 2 Exemplary assays for detecting ASR resistance. Marker SEQ ID NOSEQ ID NO SEQ ID SNP Forward Reverse SEQ ID NO SEQ ID NO Marker NOPosition Primer Primer Probe 1 Probe 2 NS0095012 1 99 9 10 25 26NS0119675 2 53 11 12 27 28 NS0102630 3 186 13 14 29 30 NS0093385 4 10915 16 31 32 NS0118716 5 366 17 18 33 34 NS0127833 6 57 19 20 35 36NS0113966 7 332 21 22 37 38 NS0118536 8 286 23 24 39 40

TABLE 3 Oligonucleotide Hybridization Probes Marker SNP HybridizationProbe SEQ Allele Marker  SEQ ID NO Position Probe ID NO DetectedNS0095012 1 99 CTGGCTTTGTGGGGCA 41 Resistance NS0095012 1 99CTGGCTTTTTGGGGCA 42 Susceptibility NS0119675 2 53 TCCTCTGAACATACTG 43Resistance NS0119675 2 53 TCCTCTGATCATACTG 44 Susceptibility NS0102630 3186 CAAGTGATCTTGAGAG 45 Resistance NS0102630 3 186 CAAGTGATATTGAGAG 46Susceptibility NS0093385 4 109 CTCACCTTTAGTTACA 47 Resistance NS00933854 109 CTCACCTTCAGTTACA 48 Susceptibility NS0118716 5 366CTACAGACCCTATGTG 49 Resistance NS0118716 5 366 CTACAGACTCTATGTG 50Susceptibility NS0127833 6 57 CATGCTAGTGTATCAG 51 Resistance NS0127833 657 CATGCTAGCGTATCAG 52 Susceptibility NS0113966 7 332 GATGCATAGAGATTGA53 Resistance NS0113966 7 332 GATGCATAAAGATTGA 54 SusceptibilityNS0118536 8 286 ATACTAAACGGCTGGT 55 Resistance NS0118536 8 286ATACTAAATGGCTGGT 56 Susceptibility

Example 4 Oligonucleotide Probes Useful for Detecting Soybean Plantswith ASR Resistance Loci by Single Base Extension Methods

Oligonucleotides can also be used to detect or type the polymorphismsassociated with ASR resistance disclosed herein by single base extension(SBE)-based SNP detection methods. Exemplary oligonucleotides for use inSBE-based SNP detection are provided in Table 3. SBE methods are basedon extension of a nucleotide primer that is hybridized to sequencesadjacent to a polymorphism to incorporate a detectable nucleotideresidue upon extension of the primer. It is also anticipated that theSBE method can use three synthetic oligonucleotides. Two of theoligonucleotides serve as PCR primers and are complementary to thesequence of the locus which flanks a region containing the polymorphismto be assayed. Exemplary PCR primers that can be used to typepolymorphisms disclosed in this invention are provided in Table 4 in thecolumns labeled “Forward Primer SEQ ID” and “Reverse Primer SEQ ID.”Following amplification of the region containing the polymorphism, thePCR product is hybridized with an extension primer which anneals to theamplified DNA adjacent to the polymorphism. DNA polymerase and twodifferentially labeled dideoxynucleoside triphosphates are thenprovided. If the polymorphism is present on the template, one of thelabeled dideoxynucleoside triphosphates can be added to the primer in asingle base chain extension. The allele present is then inferred bydetermining which of the two differential labels was added to theextension primer. Homozygous samples will result in only one of the twolabeled bases being incorporated and thus only one of the two labelswill be detected. Heterozygous samples have both alleles present, andwill thus direct incorporation of both labels (into different moleculesof the extension primer) and thus both labels will be detected.

TABLE 4Probes (extension primers) for Single Base Extension (SBE) assays.Marker SBE Probe Forward Reverse SEQ ID SNP Resistance SEQ ID Primer SEQPrimer SEQ Marker NO Position Probe (SBE) allele NO ID NO ID NONS0095012 1 99 TGTCAAATGCTGGCTTT G 57 9 10 NS0095012 1 99AATTGGAATTTGCCCCA C 58 9 10 NS0119675 2 53 ATTACCAAATCCTCTGA A 59 11 12NS0119675 2 53 TTAGAAGACCCAGTATG T 60 11 12 NS0102630 3 186AGAGAAACTCAAGTGAT C 61 13 14 NS0102630 3 186 CACATACTCACTCTCAA G 62 1314 NS0093385 4 109 ATTTAAAGACTCACCTT T 63 15 16 NS0093385 4 109TTAAACTTGGTGTAACT T 64 15 16 NS0118716 5 366 TGACACTAGCTACAGAC C 65 1718 NS0118716 5 366 ATTCTCACCTCACATAG C 66 17 18 NS0127833 6 57ACCATGAGTCATGCTAG T 67 19 20 NS0127833 6 57 TTGAATTTCCCTGATAC T 68 19 20NS0113966 7 332 GTTCTTGAAGATGCATA G 69 21 22 NS0113966 7 332CATCAACTACTCAATCT G 70 21 22 NS0118536 8 286 CAGAACAAAATACTAAA C 71 2324 NS0118536 8 286 AAATTCCAGAACCAGCC C 72 23 24

Example 5 Incorporation of Markers into Screening of Candidate SoybeanLines

ASR is an aggressive pathogen and can evolve rapidly. ASR has overcomesingle resistance genes in South America and China. Therefore, it iscritical to stack resistance genes to provide broader more durableresistance. Novel resistance sources must be identified to facilitatestacking resistance loci. Numerous resistance PIs have been identifiedfrom over 5000 PIs from Japan, China, Vietnam, and Indonesia screenedfor ASR resistance by the detached leaf technique (U.S. application Ser.No. 11/805,667). The resistant germplasm must be prioritized for furthercharacterization, such as molecular mapping

To prioritize research efforts, novel resistant PIs screened forpreviously identified ASR resistance loci Rpp1, Rpp2, Rpp3 and Rpp4.Utilizing the diagnostic SNPs associated with the Rpp genes (U.S. Appl11805667), DNA from leaf tissue of novel resistant PIs were analyzed todetermine if these PIs contain haplotypes corresponding to Rpp1, Rpp2,Rpp3 and Rpp4. PIs with a haplotype which does not correlate with Rpp1,Rpp2, Rpp3 and Rpp4, and which have a favorable phenotype with respectto any Phakopsora isolate or any geography are given priority forpopulation development and molecular mapping of the resistant loci. Inaddition, for specific geographies or specific Phakopsora isolates, PIswith a haplotype matching that of a characterized Rpp source associatedwith an uncharacterized region having a favorable phenotype, arelikewise given priority for population development and mapping of theuncharacterized resistance determinant(s). Rapid screening for knownhaplotypes thus has the advantage of minimizing effort expended on thebioassay of candidates likely to comprise just previously characterizedloci and allowing effort to be focused on novel candidates notpreviously characterized. One of skill in the art will immediately seethat the haplotype screening process can include any known haplotypeassociated with a characterized resistance phenotype and not linked toan uncharacterized resistance region, including those haplotypescomprising markers disclosed in the present invention.

Example 6 Cross-Testing of Germplasm for Differential Responses to U.S.and Ex-U.S. races of Phakopsora

The ASR resistance loci and markers of the present invention are usefulfor cross-testing haplotypes for differential associations withresistance to different races of Phakopsora. The previously describedRpp resistance genes confer less resistance against South Americanisolates of Phakopsora as compared to North American isolates; however,LG C2 appears to have a favorable haplotype near NS0137477 (AA, SEQ ID90 in U.S. Appl 11805667) which confirms tolerance to South AmericanPhakopsora isolates. Therefore, PIs which have the favorable allele atthis SNP or contain rare SNP alleles in the distal region of LG C2 aregiven priority for international testing in Brazil and other geographieswith ASR disease pressure around the world. Additionally, PIs found tobe resistant to North American isolates of Phakopsora are given priorityfor international testing over PIs lacking resistance to North Americanisolates of Phakopsora, although all are eventually tested to ensurethat a possible source of resistance, tolerance or immunity to anyex-U.S. isolates of Phakopsora is not missed. As international testingof these PIs is completed, resistance profiles with respect toPhakopsora isolates around the world are developed, and molecularmarkers and haplotypes of the present invention provide the basis fordescribing the importance of the corresponding PIs as well as relatedPIs on a regional basis. PIs which have favorable phenotypes in a givenlocale are utilized in population development and mapping efforts toidentify molecular markers associated with the resistance phenotype.

Example 7 Accumulation of Multiple Resistance Loci into SoybeanGermplasm

In other aspects, the methods and compositions of the present inventionare useful for the accumulation of multiple resistance loci intoindividual lines. In a preferred embodiment of the invention,populations are generated comprising one or more resistance loci fromnovel sources of ASR resistance that are introgressed in preferredgenetic backgrounds for testing in both North America and South America.Once ASR-resistant populations have been developed, with ASR resistanceloci fixed and agronomically elite genetics selected for viabackcrossing, near isogenic lines (NILs) are evaluated domestically andinternationally. Simultaneously, NILs with unique ASR resistancehaplotypes are intercrossed and/or forward bred to stack two or morefavorable ASR alleles. These single and stacked combinations enable thedevelopment of customized and durable ASR-resistant varieties for agiven geographic region. Single NILs also serve as differentials whenPhakopsora isolates change in a given region and provide insight towhich resistance source(s) should be deployed next. The use of markersof the present invention towards these ends will be obvious to oneskilled in the art. For example, two resistant lines (e.g., PI291309Cand PI507009) or lines with ASR resistance comprising ASR resistancelocus 14, ASR resistance locus 15, and ASR resistance locus 16, are thedonor parents for three ASR resistance loci, selected from the groupcomprising ASR resistance locus 14, ASR resistance locus 15 and ASRresistance locus 16, and monitored by screening with the molecularmarkers denoted by SEQ IDS: 1-8 and selecting for lines carrying theresistance allele for one or more of said molecular markers,representing one or more of said ASR resistance loci, wherein the SNPmarker for ASR resistance locus 14 is selected from the group consistingof NS0095012 (SEQ ID 1), NS0119675 (SEQ ID 2) and NS0102630 (SEQ ID 3)and the resistance allele for each marker is indicated in Table 3. Also,one or more SNP markers for ASR resistance locus 15 is selected from thegroup consisting of NS0093385 (SEQ ID 4), NS0118716 (SEQ ID 5) andNS0127833 (SEQ ID 6) and the resistance allele for each marker isindicated in Table 3. Also, one or more SNP markers for ASR resistancelocus 16 is selected from the group consisting of NS0113966 (SEQ ID 7)and NS0118536 (SEQ ID 8) and the resistance allele for each marker isindicated in Table 3. It will be evident to one skilled in the art thatsuch methods can moreover be used to combine one or more resistance lociof the present invention with one or more resistance loci known to theart.

The introgression of one or more resistance loci is achieved viarepeated backcrossing to a recurrent parent accompanied by selection toretain one or more ASR resistance loci from the donor parent using theabove-described markers. This backcross procedure is implemented at anystage in varietal development and occurs in conjunction with breedingfor superior agronomic characteristics or one or more traits ofinterest, including transgenic and nontransgenic traits.

Alternatively, a forward breeding approach is employed wherein one ormore ASR resistance loci can be monitored for successful introgressionfollowing a cross with a susceptible parent with subsequent generationsgenotyped for one or more ASR resistance loci and for one or moreadditional traits of interest, including transgenic and nontransgenictraits.

All patent and non-patent documents cited in this specification areincorporated herein by reference in their entireties, to the same extentas if each individual was specifically and individually indicated to beincorporated by reference. Documents cited herein as being availablefrom the World Wide Web at certain internet addresses are alsoincorporated herein by reference in their entireties. Certain biologicalsequences referenced herein by their “NCBI Accession Number” can beaccessed through the National Center of Biotechnology Information on theWorld Wide Web at ncbi.nlm.nih.gov.

As various modifications could be made in the methods herein describedand illustrated without departing from the scope of the invention, it isintended that all matter contained in the foregoing description or shownin the accompanying drawings shall be interpreted as illustrative ratherthan limiting. Having illustrated and described the principles of thepresent invention, it should be apparent to persons skilled in the artthat the invention can be modified in arrangement and detail withoutdeparting from such principles. All such modifications in arrangementand detail are considered to fall within the spirit and scope of theappended claims. Thus, the breadth and scope of the present inventionshould not be limited by any of the above-described exemplaryembodiments, but should be defined only in accordance with the followingclaims appended hereto and their equivalents.

1. A method for producing an Asian Soybean Rust (ASR) resistant soybeanplant comprising: a. performing marker assisted selection to identify asoybean plant possessing ASR resistance locus 14, wherein ASR resistancelocus 14 is obtainable from PI291309C; and b. generating a progeny ofsaid soybean plant wherein said progeny possesses said ASR resistancelocus 14 and exhibits at least partial resistance to ASR.
 2. The methodof claim 1, wherein said ASR resistance locus 14 is identifiable by oneor more of the markers selected from the group consisting of NS0095012,NS0119675, and NS0102630.
 3. The method of claim 1, wherein said markerassisted selection is conducted using an assay selected from the groupconsisting of single base extension (SBE), allele-specific primerextension sequencing (ASPE), DNA sequencing, RNA sequencing,microarray-based analyses, universal PCR, allele specific extension,hybridization, mass spectrometry, ligation, extension-ligation, and FlapEndonuclease-mediated assays.
 4. A method for producing an Asian SoybeanRust (ASR) resistant soybean plant comprising: a. performing markerassisted selection to identify a soybean plant possessing ASR resistancelocus 15, wherein ASR resistance locus 15 is obtainable from PI507009;and b. generating a progeny of said soybean plant wherein said progenypossesses said ASR resistance locus 14 and exhibits at least partialresistance to ASR.
 5. The method of claim 4, wherein said ASR resistancelocus 15 is identifiable by one or more of the markers selected from thegroup consisting of NS0093385, NS0118716, and NS0127833.
 6. The methodof claim 4, wherein said marker assisted selection is conducted using anassay selected from the group consisting of single base extension (SBE),allele-specific primer extension sequencing (ASPE), DNA sequencing, RNAsequencing, microarray-based analyses, universal PCR, allele specificextension, hybridization, mass spectrometry, ligation,extension-ligation, and Flap Endonuclease-mediated assays.
 7. A methodfor producing an Asian Soybean Rust (ASR) resistant soybean plantcomprising: a. performing marker assisted selection to identify asoybean plant possessing ASR resistance locus 16, wherein ASR resistancelocus 16 is obtainable from PI507009; and b. generating a progenysoybean plant through marker assisted breeding wherein said soybeanplant comprises ASR resistance locus 16 and at least one of ASRresistance loci selected from the group consisting of 1-15 and saidprogeny soybean plant exhibits at least partial resistance to ASR. 8.The method of claim 7, wherein said ASR resistance locus 16 isidentifiable by one or both of the markers NS0113966 and NS0118536.
 9. Asoybean plant, or a part thereof, exhibiting at least partial resistanceto ASR produced by the method of claim
 1. 10. The soybean plant, or apart thereof, of claim 9, wherein said soybean plant exhibits at leastpartial resistance to at least one race of an ASR-inducing fungus. 11.The soybean plant, or a part thereof, of claim 9, wherein saidASR-inducing fungus is Phakopsora pachyrhizi or Phakopsora meibomiae.12. A substantially purified nucleic acid molecule for the detection ofloci related to Asian Soybean Rust (ASR) resistance comprising a nucleicacid sequence selected from the group consisting of SEQ ID NO: 1 throughSEQ ID NO: 80, and complements thereof.
 13. A soybean plant, or a partthereof, exhibiting at least partial resistance to ASR produced by themethod of claim
 4. 14. A soybean plant, or a part thereof, exhibiting atleast partial resistance to ASR produced by the method of claim
 7. 15.The part of a soybean plant of claim 9, wherein said part is selectedfrom the group consisting of a seed, endosperm, ovule, pollen, stem,cutting, cell, protoplast, and tissue culture.
 16. The part of a soybeanplant of claim 13, wherein said part is selected from the groupconsisting of a seed, endosperm, ovule, pollen, stem, cutting, cell,protoplast, and tissue culture.
 17. The part of a soybean plant of claim14, wherein said part is selected from the group consisting of a seed,endosperm, ovule, pollen, stem, cutting, cell, protoplast, and tissueculture.